| 1. |
- Abelein, Axel, et al.
(författare)
-
Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly
- 2012
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 126-137
-
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.
|
|
| 2. |
- Abou-Hachem, Maher, et al.
(författare)
-
Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
- 2002
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
-
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.
|
|
| 3. |
- Adase, Christopher A., et al.
(författare)
-
Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the Tar(Ec) Chemoreceptor
- 2013
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:16, s. 2729-2738
-
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.
|
|
| 4. |
- Adase, Christopher A., et al.
(författare)
-
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
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:9, s. 1925-1932
-
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.
|
|
| 5. |
- Adrait, Annie, et al.
(författare)
-
EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse
- 2002
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:20, s. 6510-6516
-
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.
|
|
| 6. |
- Aguilar, Ximena, 1978-, et al.
(författare)
-
Macromolecular crowding extended to a heptameric system : the co-chaperonin protein 10
- 2011
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:14, s. 3034-3044
-
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.
|
|
| 7. |
- Ahl, Ing-Marie, et al.
(författare)
-
Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry
- 2009
-
Ingår i: BIOCHEMISTRY. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:41, s. 9932-9940
-
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
|
|
| 8. |
|
|
| 9. |
- Ali, Muhammad, 1990-, et al.
(författare)
-
Elucidation of Short Linear Motif-Based Interactions of the FERM Domains of Ezrin, Radixin, Moesin, and Merlin
- 2023
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 62:11, s. 1594-1607
-
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.
|
|
| 10. |
- Alkemar, Gunnar, et al.
(författare)
-
Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA
- 2006
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:26, s. 8067-8078
-
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.
|
|
