| 1. |
- Abelein, Axel, et al.
(författare)
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Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 126-137
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Tidskriftsartikel (refereegranskat)abstract
- The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.
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| 2. |
- Abou-Hachem, Maher, et al.
(författare)
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Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
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Tidskriftsartikel (refereegranskat)abstract
- Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
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| 3. |
- Adase, Christopher A., et al.
(författare)
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Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the Tar(Ec) Chemoreceptor
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:16, s. 2729-2738
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Tidskriftsartikel (refereegranskat)abstract
- Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia colt aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild type Tar(Ec) is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six residue sequence at positions 207-212 Moving various combinations of aromatic residues among these positions generates substantial changes M receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.
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| 4. |
- Adase, Christopher A., et al.
(författare)
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The Residue Composition of the Aromatic Anchor of the Second Transmembrane Helix Determines the Signaling Properties of the Aspartate/Maltose Chemoreceptor Tar of Escherichia coli
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:9, s. 1925-1932
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Tidskriftsartikel (refereegranskat)abstract
- Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was: present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.
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| 5. |
- Adrait, Annie, et al.
(författare)
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EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:20, s. 6510-6516
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the μ-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.
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| 6. |
- Aguilar, Ximena, 1978-, et al.
(författare)
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Macromolecular crowding extended to a heptameric system : the co-chaperonin protein 10
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:14, s. 3034-3044
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Tidskriftsartikel (refereegranskat)abstract
- Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.
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| 7. |
- Ahl, Ing-Marie, et al.
(författare)
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Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry
- 2009
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Ingår i: BIOCHEMISTRY. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:41, s. 9932-9940
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular superoxide dismutase (ECSOD) interacts with heparin through its C-terminal domain. In this study we used isothermal titration calorimetry (ITC) to get detailed thermodynamic information about the interaction. We have shown that the interaction between ECSOD and intestinal mucosal heparin (M-w 6000-30000 Da) is exothermic and driven by enthalpy at physiological salt concentration. However, the contribution from entropy is favorable for binding or small isolated heparin fragments. By studying different size-defined heparin fragments, we also concluded that it hexasaccharide moiety is sufficient for strong binding to ECSOD. The binding involves proton transfer from the buffer to the ECSOD-heparin complex, and the results indicate that the number of ionic interactions made between ECSOD and heparin upon binding varies from three to five for heparin and an octasaccharide fragment, respectively. Surprisingly and despite the many charges found oil both the protein and the polysaccharide, our results indicate that the nonionic contribution to the binding is large. From the temperature dependence we have calculated the constant pressure heat capacity change (Delta C-p) of the interaction to -644 J K-1 mol(-1) and -306 J K-1 mol(-1) for heparin and all octasaccharide, respectively
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| 8. |
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| 9. |
- Ali, Muhammad, 1990-, et al.
(författare)
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Elucidation of Short Linear Motif-Based Interactions of the FERM Domains of Ezrin, Radixin, Moesin, and Merlin
- 2023
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 62:11, s. 1594-1607
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Tidskriftsartikel (refereegranskat)abstract
- The ERM (ezrin, radixin,and moesin) family of proteins and therelated protein merlin participate in scaffolding and signaling eventsat the cell cortex. The proteins share an N-terminal FERM [band four-point-one(4.1) ERM] domain composed of three subdomains (F1, F2, and F3) withbinding sites for short linear peptide motifs. By screening the FERMdomains of the ERMs and merlin against a phage library that displayspeptides representing the intrinsically disordered regions of thehuman proteome, we identified a large number of novel ligands. Wedetermined the affinities for the ERM and merlin FERM domains interactingwith 18 peptides and validated interactions with full-length proteinsthrough pull-down experiments. The majority of the peptides containedan apparent Yx-[FILV] motif; others show alternative motifs. We defineddistinct binding sites for two types of similar but distinct bindingmotifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computationalpeptide docking protocols and mutational analysis. We provide a detailedmolecular understanding of how the two types of peptides with distinctmotifs bind to different sites on the moesin FERM phosphotyrosinebinding-like subdomain and uncover interdependencies between the differenttypes of ligands. The study expands the motif-based interactomes ofthe ERMs and merlin and suggests that the FERM domain acts as a switchableinteraction hub.
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| 10. |
- Alkemar, Gunnar, et al.
(författare)
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Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA
- 2006
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:26, s. 8067-8078
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Tidskriftsartikel (refereegranskat)abstract
- Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.
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| 11. |
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| 12. |
- Almlöf, Martin, et al.
(författare)
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Energetics of codon-anticodon recognition on the small ribosomal subunit
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:1, s. 200-209
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Tidskriftsartikel (refereegranskat)abstract
- Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.
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| 13. |
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| 14. |
- Almstedt, Karin, et al.
(författare)
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Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
- 2009
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:23, s. 5358-5364
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Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.
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| 15. |
- Almstedt, Karin, 1980-, et al.
(författare)
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Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
- 2008
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Ingår i: Biochemistry. - Washington : ACS. - 0006-2960 .- 1520-4995. ; 47:5, s. 1288-1298
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Tidskriftsartikel (refereegranskat)abstract
- Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.
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| 16. |
- Andér, Martin, et al.
(författare)
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Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?
- 2009
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Ingår i: Biochemistry. - Washington, DC, USA : American Chemical Society. - 0006-2960 .- 1520-4995. ; 48:15, s. 3483-3489
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Tidskriftsartikel (refereegranskat)abstract
- The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.
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| 17. |
- Andersson, D., et al.
(författare)
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Cofactor-induced refolding : Refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)
- 2001
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:9, s. 2653-2661
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Tidskriftsartikel (refereegranskat)abstract
- The stability versus unfolding to the molten globule intermediate of bovine carbonic anhydrase II (BCA II) in guanidine hydrochloride (GuHCl) was found to depend on the metal ion cofactor [Zn(II) or Co(II)], and the apoenzyme was observed to be least stable. Therefore, it was possible to find a denaturant concentration (1.2 M GuHCl) at which refolding from the molten globule to the native state could be initiated merely by adding the metal ion to the apo molten globule. Thus, refolding could be performed without changing the concentration of the denaturant. The molten globule intermediate of BCA II could still bind the metal cofactor. Cofactor-effected refolding from the molten globule to the native state can be summarized as follows: (1) initially, the metal ion binds to the molten globule, (2) compaction of the metal-binding site region is then induced by the metal ion binding, (3) a functioning active center is formed, and (4) finally, the native tertiary structure is generated in the outer parts of the protein.
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| 18. |
- André, Ingemar, et al.
(författare)
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Streptococcal M protein: Structural studies of the hypervariable region, free and bound to human C4BP
- 2006
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:14, s. 4559-4568
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an similar to 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of similar to 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.
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| 19. |
- Antzutkin, Oleg, et al.
(författare)
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Supramolecular structural constraints on Alzheimer's β-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:51, s. 15436-15450
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Tidskriftsartikel (refereegranskat)abstract
- We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1-42) and by residues 10-35 of the full-length peptide (Aβ10-35). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of β-sheets present in the characteristic amyloid cross-β structural motif and the assembly of these β-sheets into a fibril. EM images of negatively stained Aβ10-35 fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Aβ10-35 fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-β layer. Aβ10-35 fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Aβ1-42 fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-β layer are also formed by Aβ1-42 and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Aβ10-35 fibrils, using multiple-quantum 13C NMR, 13C-13C dipolar recoupling, and 15N-13C dipolar recoupling techniques, support the in-register parallel β-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this β-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Aβ1-42 fibrils, which represent the first NMR data on Aβ1-42 fibrils, also indicate an in-register parallel β-sheet organization. These results, along with previously reported data on Aβ1-40 fibrils, suggest that the supramolecular structures of Aβ10-35, Aβ1-40, and Aβ1-42 fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.
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| 20. |
- Apostolov, Rossen, et al.
(författare)
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Membrane attachment facilitates ligand access to the active site in monoamine oxidase A
- 2009
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:25, s. 5864-5873
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Tidskriftsartikel (refereegranskat)abstract
- Monoamine oxidase membrane enzymes are responsible for the catalytic breakdown of extra- and intracellular neurotransmitters and are targets for the development of central nervous system drugs. We analyzed the dynamics of rat MAOA by performing multiple independent molecular dynamics simulations of membrane-bound and membrane-free forms to clarify the relationship between the mechanics of the enzyme and its function, with particular emphasis on the significance of membrane attachment. Principal component analysis of the simulation trajectories as well as correlations in the fluctuations of the residues pointed to the existence of three domains that define the global dynamics of the protein. Interdomain anticorrelated movements in the membrane-bound system facilitated the relaxation of interactions between residues surrounding the substrate cavity and induced conformational changes which expanded the active site cavity and opened putative pathways for substrate uptake and product release. Such events were less pronounced in the membrane-free system due to differences in the nature of the dominant modes of motion. The presence of the lipid environment is suggested to assist in decoupling the interdomain motions, consistent with the observed reduction in enzyme activity under membrane-free conditions. Our results are also in accordance with mutational analysis which shows that modifications of interdomain hinge residues decrease the activity of rat MAOA in solution.
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| 21. |
- Ariöz, Candan, 1983-, et al.
(författare)
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Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:33, s. 5533-5544
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Tidskriftsartikel (refereegranskat)abstract
- Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
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| 22. |
- Awad, Wael, et al.
(författare)
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Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:7, s. 1166-1177
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.
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| 23. |
- Bai, Shi, et al.
(författare)
-
Contiguous binding of decylsulfate on the interface-binding surface of pancreatic phospholipase A2
- 2008
-
Ingår i: Biochemistry. - : ACS. - 0006-2960 .- 1520-4995. ; 47:9, s. 2899-2907
-
Tidskriftsartikel (refereegranskat)abstract
- Pig pancreatic IB phospholipase A(2) (PLA2) forms three distinguishable premicellar E-i(#) (i = 1, 2, and 3) complexes at successively higher decylsulfate concentrations. The Hill coefficient for E-1(#) is n(1) = 1.6, and n(2) and n(3) for E-2(#) and E-3(#) are about 8 each. Saturation-transfer difference nuclear magnetic resonance (NMR) and other complementary results with PLA2 show that decylsulfate molecules in E-2(#) and E-3(#) are contiguously and cooperatively clustered on the interface-binding surface or i-face that makes contact with the substrate interface. In these complexes, the saturation-transfer difference NMR signatures of H-1 in decylsulfate are different. The decylsulfate epitope for the successive E, complexes increasingly resembles the micellar complex formed by the binding of PLA2 to preformed micelles. Contiguous cooperative amphiphile binding is predominantly driven by the hydrophobic effect with a modest electrostatic shielding of the sulfate head group in contact with PLA2. The formation of the complexes is also associated with structural change in the enzyme. Calcium affinity of E-2(#) appears to be modestly lower than that of the free enzyme and Ell. Binding of decylsulfate to the i-face does not require the catalytic calcium required for the substrate binding to the active site and for the chemical step. These results show that E-i(#) complexes are useful to structurally characterize the cooperative sequential and contiguous binding of amphiphiles on the i-face. We suggest that the allosteric changes associated with the formation of discrete E-i(#) complexes are surrogates for the catalytic and allosteric states of the interface activated PLA2.
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| 24. |
- Balaz, Martina, et al.
(författare)
-
Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue
- 2007
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:24, s. 7233-7251
-
Tidskriftsartikel (refereegranskat)abstract
- Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.
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| 25. |
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| 26. |
- Balbach, John J., et al.
(författare)
-
Amyloid Fibril Formation by Aβ16-22, a Seven-Residue Fragment of the Alzheimer's β-Amyloid Peptide, and Structural Characterization by Solid State NMR
- 2000
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 39:45, s. 13748-13759
-
Tidskriftsartikel (refereegranskat)abstract
- The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2, called Aβ16-22 and representing residues 16-22 of the full-length β-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in Aβ16-22 fibrils are investigated by solid state 13C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a β-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit 13C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional 13C-13C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain 13C NMR signals and indicates that the β-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. 13C multiple-quantum (MQ) NMR and 13C/15N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of β-sheets in the Aβ16-22 fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of β-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. Aβ16-22 is among the shortest fibril-forming fragments of full-length β-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.
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| 27. |
- Balogh, Larissa M., et al.
(författare)
-
Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals
- 2009
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:32, s. 7698-7704
-
Tidskriftsartikel (refereegranskat)abstract
- The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 angstrom resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha 9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the 0 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, Are cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal Closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.
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| 28. |
- Balogh, Larissa M., et al.
(författare)
-
Substrate Specificity Combined with Stereopromiscuity in Glutathione Transferase A4-4-Dependent Metabolism of 4-Hydroxynonenal
- 2010
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:7, s. 1541-1548
-
Tidskriftsartikel (refereegranskat)abstract
- Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and ail engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward ail endogenously formed toxin.
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| 29. |
- Bauer, Mikael, et al.
(författare)
-
Calmodulin Binding to the Polybasic C-Termini of STIM Proteins Involved in Store-Operated Calcium Entry.
- 2008
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:23, s. 6089-6091
-
Tidskriftsartikel (refereegranskat)abstract
- Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D
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| 30. |
- Bavdek, Andrej, et al.
(författare)
-
Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O.
- 2007
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:14, s. 4425-4437
-
Tidskriftsartikel (refereegranskat)abstract
- Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.
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| 31. |
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| 32. |
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| 33. |
- Behnen, Petra, et al.
(författare)
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Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes.
- 2012
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:34, s. 6718-6727
-
Tidskriftsartikel (refereegranskat)abstract
- Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.
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| 34. |
- Beke-Somfai, Tamas, 1977, et al.
(författare)
-
Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites
- 2010
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 49:3, s. 401-403
-
Tidskriftsartikel (refereegranskat)abstract
- Despite exhaustive chemical and crystal structure studies, the mechanistic details of how F o F 1 -ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO 3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in β TP and, thus, promote ATP. © 2009 American Chemical Society.
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| 35. |
- Belogurov, G A, et al.
(författare)
-
Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase
- 2000
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 39:45, s. 13931-13938
-
Tidskriftsartikel (refereegranskat)abstract
- Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.
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| 36. |
- Belogurov, Georgiy A, et al.
(författare)
-
Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity
- 2005
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:6, s. 2088-2096
-
Tidskriftsartikel (refereegranskat)abstract
- Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.
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| 37. |
- Benach, J, et al.
(författare)
-
Structure of bacterial 3 beta/17 beta-hydroxysteroid dehydrogenase at 1.2 angstrom resolution : A model for multiple steroid recognition
- 2002
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:50, s. 14659-14668
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Conramonas testosteroni. It catalyzes the reversible reduction/ dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 Angstrom resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.
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| 38. |
- Berg, Otto, et al.
(författare)
-
Cooperative binding of monodisperse anionic amphiphiles to the i-Face : Phospholipase A2-paradigm for interfacial binding
- 2004
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:25, s. 7999-8013
-
Tidskriftsartikel (refereegranskat)abstract
- Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A2 (PLA2) to form the premicellar complexes (Ei#) are characterized to discern the short-range specific interactions. Typically, Ei# complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (Ei#, i = 1, 2, 3). In the presence of calcium, the dissociation constant K1 for the E1# complex of PLA2 with decyl sulfate (CMC = 4500 μM) is 70 μM with a Hill coefficient n1 = 2.1 ± 0.2; K2 for E2# is 750 μM with n2 = 8 ± 1, and K3 for E3# is 4000 μM with an n3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of Ei#; (c) Ki and ni do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the Ei# complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in Ei#. The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of Ei#. We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and Ei# aggregates is discussed.
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| 39. |
- Berg, Otto G., et al.
(författare)
-
Thermodynamic Reciprocity of the Inhibitor Binding to the Active Site and the Interface Binding Region of IB Phospholipase A2
- 2009
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:14, s. 3209-3218
-
Tidskriftsartikel (refereegranskat)abstract
- Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E-i(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes ((E) over tilde (i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the (E) over tilde I-i complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the (E) over tilde (i) complexes with PCU are qualitatively similar to those of E-i(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E-2(#) complex or, equivalently, the shift in the E-1(#) to E-2(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.
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| 40. |
- Berg, Otto, et al.
(författare)
-
Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine
- 2004
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:7, s. 2080-2090
-
Tidskriftsartikel (refereegranskat)abstract
- Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E# with monodisperse diheptanoylphosphatidylcholine (DC7PC) that is distinguishable from the E* complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC7PC give an apparent dissociation constant of K2 = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E# are distinct from those of the E* complex. The aggregation and partitioning behaviors suggest that the acyl chains in E# but not in E* remain exposed to the aqueous phase. The free (E) and complexed (E# and E*) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC7PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis−Menten complex formed with monodisperse DC7PC is likely to be E#S or its aggregate rather than the classical monodisperse ES complex.
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| 41. |
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| 42. |
- Bernat, Gabor, et al.
(författare)
-
pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution.
- 2002
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5830-5843
-
Tidskriftsartikel (refereegranskat)abstract
- We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH <4.8). Therefore, we refrain from providing error limits for the determined pK's. Our results indicate that the S1 --> S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC.
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| 43. |
- Berndt, Kurt D, et al.
(författare)
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Designed replacement of an internal hydration water molecule in BPTI : structural and functional implications of a glycine-to-serine mutation.
- 1993
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 32, s. 4564-4570
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Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.
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| 44. |
- Billsten, Helena, et al.
(författare)
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Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:12, s. 4127-4136
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Tidskriftsartikel (refereegranskat)abstract
- LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with I I saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S, level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S I energy of lycopene in n-hexane was determined to be similar to12 500 +/- 150 cm(-1), by direct measurement of the S-1-S-2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S, energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischian1l7n, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is similar to80% in lyco-LH2 and similar to95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S-1 --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt Si energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S, state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S-1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B 800 Q, state, leaving only the Crt S-1 --> B 850 channel, strongly suggesting that Crt S-1 --> BChl energy transfer is controlled by the relative Crt S-1 and BChl Q(y) energies.
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| 45. |
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| 46. |
- Bjelic, Sinisa, et al.
(författare)
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Cold adaptation of enzyme reaction rates
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:38, s. 10049-10057
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Tidskriftsartikel (refereegranskat)abstract
- A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.
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| 47. |
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| 48. |
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| 49. |
- Bjork, Ingemar, et al.
(författare)
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The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases
- 1996
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:33, s. 10720-10726
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Tidskriftsartikel (refereegranskat)abstract
- The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied were due almost exclusively to increased dissociation rate constants. The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity indicated that the first hairpin loop (the "QVVAG-region"), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases.
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| 50. |
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