| 1. |
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
- Berndt, Kurt D, et al.
(författare)
-
Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures
- 1992
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 227, s. 757-775
-
Tidskriftsartikel (refereegranskat)abstract
- A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
- Byström, Anders S, et al.
(författare)
-
Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species
- 1989
-
Ingår i: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 208:4, s. 575-586
-
Tidskriftsartikel (refereegranskat)abstract
- The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.
|
|
| 10. |
|
|
| 11. |
- Emanuelsson, Olof, et al.
(författare)
-
In silico prediction of the peroxisomal proteome in fungi, plants and animals.
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:2, s. 443-456
-
Tidskriftsartikel (refereegranskat)abstract
- In an attempt to improve our abilities to predict peroxisomal proteins, we have combined machine-learning techniques for analyzing peroxisomal targeting signals (PTS1) with domain-based cross-species comparisons between eight eukaryotic genomes. Our results indicate that this combined approach has a significantly higher specificity than earlier attempts to predict peroxisomal localization, without a loss in sensitivity. This allowed us to predict 430 peroxisomal proteins that almost completely lack a localization annotation. These proteins can be grouped into 29 families covering most of the known steps in all known peroxisomal pathways. In general, plants have the highest number of predicted peroxisomal proteins, and fungi the smallest number.
|
|
| 12. |
|
|
| 13. |
- Hughes, Diarmaid, 1956-
(författare)
-
Both genes for EF-Tu in Salmonella typhimurium are individually dispensible for growth
- 1990
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 215:1, s. 41-51
-
Tidskriftsartikel (refereegranskat)abstract
- Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.
|
|
| 14. |
- Kirsebom, Leif A, et al.
(författare)
-
Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors
- 1988
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 204:4, s. 879-888
-
Tidskriftsartikel (refereegranskat)abstract
- We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.
|
|
| 15. |
- Kirsebom, Leif A, et al.
(författare)
-
Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates
- 1989
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 207:4, s. 837-840
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.
|
|
| 16. |
|
|
| 17. |
|
|
| 18. |
|
|
| 19. |
|
|
| 20. |
- Lundin, Cecilia, et al.
(författare)
-
RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells
- 2003
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836. ; 328:3, s. 521-35
-
Tidskriftsartikel (refereegranskat)abstract
- The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than γ-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.
|
|
| 21. |
- Malisauskas, Mantas, et al.
(författare)
-
Amyloid protofilaments from the calcium-binding protein equine lysozyme : formation of ring and linear structures depends on pH and metal ion concentration
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:4, s. 879-890
-
Tidskriftsartikel (refereegranskat)abstract
- The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 °C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca2+ the protofilaments are present as annular structures with a diameter of 40–50 nm. In the presence of 10 mM CaCl2 the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 °C and 57 °C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70–80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1–80 and 54–125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as -synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.
|
|
| 22. |
- Masson, Patrick, et al.
(författare)
-
Drosophila Proteasome Regulator REGγ : Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 327:5, s. 1001-1012
-
Tidskriftsartikel (refereegranskat)abstract
- The proteasome regulator REG (PA28γ) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGγ in metazoan cells is currently unknown. To understand the role of Drosophila REGγ we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGγ transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGγ transcription. To confirm that REGγ has a role in cell cycle progression, the effect of removing REGγ from S2 cells was tested using RNA interference. Drosophila cells depleted of REGγ showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGγ is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGγ was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.
|
|
| 23. |
- Meining, Winfried, et al.
(författare)
-
The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6 A resolution
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 331:5, s. 1053-1063
-
Tidskriftsartikel (refereegranskat)abstract
- Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here,we report the structure of this construct in complex with riboflavin at 2.6 Angstrom resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.
|
|
| 24. |
|
|
| 25. |
|
|
| 26. |
|
|
| 27. |
|
|
| 28. |
|
|
| 29. |
|
|
| 30. |
|
|
| 31. |
- von Ossowski, I., et al.
(författare)
-
Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 333:4, s. 817-829
-
Tidskriftsartikel (refereegranskat)abstract
- The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
|
|
| 32. |
- Wikström, P M, et al.
(författare)
-
Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli
- 1988
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 203:1, s. 141-152
-
Tidskriftsartikel (refereegranskat)abstract
- The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.
|
|
| 33. |
- Zhang, X F, et al.
(författare)
-
A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 328:1, s. 167-182
-
Tidskriftsartikel (refereegranskat)abstract
- 6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 Angstrom, 1.85 Angstrom, 2.05 Angstrom and 2.2 Angstrom, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of singlesite mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.
|
|
| 34. |
- Abdulkarim, Farhad, et al.
(författare)
-
Homologous recombination between the tuf genes of Salmonella typhimurium
- 1996
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 260:4, s. 506-522
-
Tidskriftsartikel (refereegranskat)abstract
- The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.
|
|
| 35. |
- Aboualizadeh, F., et al.
(författare)
-
Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling
- 2021
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 433:23
-
Tidskriftsartikel (refereegranskat)abstract
- Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome. By screening a library of 86 SH2 domain-containing full length proteins, 30 novel ALK interactors were identified. Many of their interactions are correlated to ALK phosphorylation activity: oncogenic ALK mutations potentiate the interactions and ALK inhibitors attenuate the interactions. Among the novel interactors, NCK2 was further verified in neuroblastoma cells using co-immunoprecipitation. Modulation of ALK activity by addition of inhibitors lead to concomitant changes in the tyrosine phosphorylation status of NCK2 in neuroblastoma cells, strongly supporting the functionality of the ALK/NCK2 interaction. Our study provides a resource list of potential novel ALK signaling components for further study. (C) 2021 The Authors. Published by Elsevier Ltd.
|
|
| 36. |
- Abu-Raya, Bahaa, et al.
(författare)
-
Antibody and B-cell Immune Responses Against Bordetella Pertussis Following Infection and Immunization
- 2023
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 435:24
-
Forskningsöversikt (refereegranskat)abstract
- Neither immunization nor recovery from natural infection provides life-long protection against Bordetella pertussis. Replacement of a whole-cell pertussis (wP) vaccine with an acellular pertussis (aP) vaccine, mutations in B. pertussis strains, and better diagnostic techniques, contribute to resurgence of number of cases especially in young infants. Development of new immunization strategies relies on a comprehensive understanding of immune system responses to infection and immunization and how triggering these immune components would ensure protective immunity. In this review, we assess how B cells, and their secretory products, antibodies, respond to B. pertussis infection, current and novel vaccines and highlight similarities and differences in these responses. We first focus on antibody-mediated immunity. We discuss antibody (sub)classes, elaborate on antibody avidity, ability to neutralize pertussis toxin, and summarize different effector functions, i.e. ability to activate complement, promote phagocytosis and activate NK cells. We then discuss challenges and opportunities in studying B-cell immunity. We highlight shared and unique aspects of B-cell and plasma cell responses to infection and immunization, and discuss how responses to novel immunization strategies better resemble those triggered by a natural infection (i.e., by triggering responses in mucosa and production of IgA). With this comprehensive review, we aim to shed some new light on the role of B cells and antibodies in the pertussis immunity to guide new vaccine development.
|
|
| 37. |
|
|
| 38. |
|
|
| 39. |
- Aisenbrey, Christopher, et al.
(författare)
-
Macromolecular Crowding at Membrane Interfaces : Adsorption and Alignment of Membrane Peptides
- 2008
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 375, s. 376-385
-
Tidskriftsartikel (refereegranskat)abstract
- Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH4, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.
|
|
| 40. |
- Alikhani, Nyosha, et al.
(författare)
-
Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species
- 2011
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 410:3, s. 400-410
-
Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.
|
|
| 41. |
- Almstedt, Karin, 1980-, et al.
(författare)
-
Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
- 2004
-
Ingår i: Journal of Molecular Biology. - Oxford : Elsevier. - 0022-2836 .- 1089-8638. ; 342:2, s. 619-633
-
Tidskriftsartikel (refereegranskat)abstract
- Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.
|
|
| 42. |
|
|
| 43. |
- Andersen, Birgit, et al.
(författare)
-
A recruited protease is involved in catabolism of pyrimidines.
- 2008
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 379:2, s. 243-250
-
Tidskriftsartikel (refereegranskat)abstract
- In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.
|
|
| 44. |
|
|
| 45. |
- Andersson, C. Evalena, et al.
(författare)
-
Activation of Ribokinase by Monovalent Cations
- 2002
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 315:3, s. 409-419
-
Tidskriftsartikel (refereegranskat)
|
|
| 46. |
- Andersson, Evalena, et al.
(författare)
-
Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme
- 2010
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 397:4, s. 1003-1016
-
Tidskriftsartikel (refereegranskat)abstract
- Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16Da, 136aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P21 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.
|
|
| 47. |
- Andersson, Magnus, et al.
(författare)
-
A structural basis for sustained bacterial adhesion : Biomechanical properties of CFA/I Pili
- 2012
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 415:5, s. 918-928
-
Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized at single organelle level the intrinsic biomechanical properties and kinetics of individual CFA/I pili, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P-pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix, and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.
|
|
| 48. |
- Andreasson, Ulrika, et al.
(författare)
-
The human IgE-encoding transcriptome to assess antibody repertoires and repertoire evolution
- 2006
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 362:2, s. 212-227
-
Tidskriftsartikel (refereegranskat)abstract
- Upon encounter with antigen, the B lymphocyte population responds by producing a diverse set of antigen-specific antibodies of various isotypes. The vast size of the responding populations makes it very difficult to study clonal evolution and repertoire composition occurring during these processes in humans. Here, we have explored an approach utilizing the H-EPSILON-encoding transcriptome to investigate aspects of repertoire diversity during the season of antigen exposure. We show through sequencing of randomly picked transcripts that the sizes of patients' repertoires are relatively small. This specific aspect of the transcriptome allows us to construct evolutionary trees pinpointing features of somatic hypermutation as it occurs in humans. Despite the small size of the repertoires, they are highly diverse with respect to VDJ gene usage, suggesting that the H-EPSILON-encoding transcriptome is a faithful mimic of other class-switched isotypes. Importantly, it is possible to use antibody library and selection technologies to define the specificity of clonotypes identified by random sequencing. The small size of the H-EPSILON-encoding transcriptome of peripheral blood B cells, the simple identification of clonally related sets of genes in this population, and the power of library and selection technologies ensure that this approach will allow us to investigate antibody evolution in human B lymphocytes of known specificity. As H-EPSILON repertoires show many of the hallmarks of repertoires encoding other isotypes, we suggest that studies of this type will have an impact on our understanding of human antibody evolution even beyond that occurring in the IgE-producing B cell population.
|
|
| 49. |
- Antonyuk, Svetlana V, et al.
(författare)
-
The structure of human extracellular copper-zinc superoxide dismutase at 1.7 A resolution : insights into heparin and collagen binding
- 2009
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 388:2, s. 310-326
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 A resolution. The overall subunit fold and the subunit-subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu-Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.
|
|
| 50. |
- Arike, Liisa, et al.
(författare)
-
The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria.
- 2016
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 428:16, s. 3221-3229
-
Forskningsöversikt (refereegranskat)abstract
- All mucins are highly O-glycosylated by variable glycans depending on species, histoblood group and organ. This makes the intestinal main mucin MUC2 non-degradable by the host digestive system but well by both commensal and pathogenic bacteria. The MUC2 glycans are important for selection of the commensal bacteria and act as a nutritional source for the bacteria; this also helps the host to recover some of the energy spent on constantly renewing the protective mucus layer. Glycosylation is the most diverse and common posttranslational modification of cell surfaces and secreted proteins. N-Glycosylation is most well studied and predictable, whereas O-glycosylation is more diverse and less well understood. O-Glycosylation is also often called mucin-type glycosylation as it is typical for mucins that often have more than 80% of the mass as O-glycans. This review will discuss the mucin-type O-glycosylation and especially the O-glycosylation of human and mice intestinal mucin MUC2 in relation to bacteria and disease.
|
|
