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1.
The field of regenerative medicine offers hope for the development of a cell-based therapy for the repair of articular cartilage (AC). Yet, the greatest challenge in the use of stem cells for tissue repair, is understanding how the cells respond to stimuli and using that knowledge to direct cell fate. Novel methods that utilize stem cells in cartilage regeneration will require specific spatio-temporal controls of the biochemical and biophysical signaling environments. Current chondrogenic differentiation research focuses on the roles of biochemical stimuli like growth factors, hormones, and small molecules, and the role of the physical environment and mechanical stimuli, such as compression and shear stress, which likely act through mechanical receptors. Numerous signals are associated with chondrogenic-like activity of cells in different systems, however many variables for a controlled method still need to be optimized; e.g., spatial and temporal application of the stimuli, and time of transplantation of an engineered construct. Understanding the necessary microenvironmental signals for cell differentiation will advance cell therapy for cartilage repair. 相似文献
2.
《Journal of molecular biology》2021,433(15):167047
In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm. 相似文献
3.
Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme
with major elements of secondary structure being β-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high
temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding.
Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified
in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted. 相似文献
4.
David M. Thomas Gregory D. Ferguson Harvey R. Herschman Lisa A. Elferink 《Molecular biology of the cell》1999,10(7):2285-2295
Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non-calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly. 相似文献
5.
Simone Fulle 《Journal of molecular biology》2009,387(2):502-2578
A sophisticated interplay between the static properties of the ribosomal exit tunnel and its functional role in cotranslational processes is revealed by constraint counting on topological network representations of large ribosomal subunits from four different organisms. As for the global flexibility characteristics of the subunit, the results demonstrate a conserved stable structural environment of the tunnel. The findings render unlikely that deformations of the tunnel move peptides down the tunnel in an active manner. Furthermore, the stable environment rules out that the tunnel can adapt widely so as to allow tertiary folding of nascent chains. Nevertheless, there are local zones of flexible nucleotides within the tunnel, between the peptidyl transferase center and the tunnel constriction, and at the tunnel exit. These flexible zones strikingly agree with previously identified folding zones. As for cotranslational elongation regulation, flexible residues in the β-hairpin of the ribosomal L22 protein were verified, as suggested previously based on structural results. These results support the hypothesis that L22 can undergo conformational changes that regulate the tunnel voyage of nascent polypeptides. Furthermore, rRNA elements, for which conformational changes have been observed upon interaction of the tunnel wall with a nascent SecM peptide, are less strongly coupled to the subunit core. Sequences of coupled rigid clusters are identified between the tunnel and some of these elements, suggesting signal transmission by a domino-like mechanical coupling. Finally, differences in the flexibility of the glycosidic bonds of bases that form antibiotics-binding crevices within the peptidyl transferase center and the tunnel region are revealed for ribosomal structures from different kingdoms. In order to explain antibiotics selectivity, action, and resistance, according to these results, differences in the degrees of freedom of the binding regions may need to be considered. 相似文献
6.
Rebeca Bocanegra Miguel?ángel Fuertes Alicia Rodríguez-Huete José?Luis Neira Mauricio?G. Mateu 《Biophysical journal》2015,108(2):338-349
Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers. 相似文献
7.
Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.The classical autotransporter (AT) family, also known as the type Va protein secretion system, represents the largest group of proteins secreted by Gram-negative bacteria and includes many virulence factors from important human pathogens (10, 17). Bacteria produce AT proteins as large polypeptide precursors, with their virulence activity (e.g., cytotoxins, adhesins, and proteases, etc.) present in a passenger domain flanked by an N-terminal signal peptide (sp) for Sec-dependent translocation across the bacterial inner membrane (IM) and a C-terminal domain of ∼30 to 40 kDa for insertion into the bacterial outer membrane (OM) (see Fig. 2A). A self-translocation model was originally proposed to explain the secretion mechanism of AT proteins across the OM, based mostly on data obtained with the IgA protease (IgAP) from Neisseria gonorrhoeae (43). In this model the C-terminal domain of ATs was supposed to fold in the OM as a β-barrel protein with an internal hydrophilic pore that could be used for the translocation of the passenger domain. The finding that the B subunit of cholera toxin (CtxB) should not have disulfide bonds for its secretion when fused as a heterologous passenger to the C-terminal domain of IgAP (30, 31) indirectly suggests passenger translocation in an unfolded conformation through a narrow channel expected for a β-barrel. Similar observations with the C-terminal domains of IcsA from Shigella flexneri (56) and AIDA-I from Escherichia coli (36) supported this model.Previous work done by our group challenged the original self-translocation model, since a 45-kDa C-terminal fragment of IgAP was shown to form oligomeric ring-shaped complexes with a central hydrophilic pore of ∼2 nm (63). In addition, this C-terminal fragment of IgAP was found to translocate folded immunoglobulin (Ig) domains with disulfide bonds to the bacterial surface, indicating that at least a ∼2-nm pore was being used for passenger secretion (61, 62). These data led us to propose a “multimeric” version of the self-translocation model in which the secretion of the passenger may occur through the central channel assembled by the oligomerization of the C-terminal domains in the OM. Studies with IcsA from S. flexneri (7, 46, 47, 64) and EspP from E. coli (53) also provided evidence indicating that native and heterologous passengers adopt folded or at least partially folded conformations in the periplasm before OM translocation. Conversely, a limited capacity for the translocation of folded native passengers with engineered disulfide bonds has been reported by studies with Hbp from E. coli (23) and pertactin from Bordetella pertussis (24). Crystallographic structures of the C-terminal domains of NalP from Neisseria meningitidis (41) and EspP from E. coli (2) revealed distinct β-barrel folding with 12 amphipathic β-strands and one N-terminal α-helix filling the central hydrophilic pore of the β-barrel. No indication of oligomerization was obtained with the crystallographic data. In addition, the putative protein-conducting channels of the EspP and NalP β-barrels (of ∼1 nm in diameter) were found to be closed due to the presence of the internal α-helix, which would impede the transport of passenger polypeptides (either folded or unfolded) through the reported structures. Thus, an alternative model was proposed for the assisted translocation of ATs (3, 41), in which the protein-conducting channel for secretion across the OM would be provided by the conserved Bam complex. The Bam complex is required for the insertion of β-barrel proteins (32), and the depletion of its essential component BamA (formerly YaeT in E. coli and Omp85 in Neisseria) prevents the insertion of several ATs in the OM (i.e., IcsA and SepA from S. flexneri, AIDA-I and Hbp from E. coli, and BrkA from B. pertussis) (21, 50). BamA was reported to form hydrophilic pores in lipid membranes in vitro (54) and to cross-link in vivo with the passenger domain of a slow-secretion mutant of EspP (19), which supports a role for BamA in translocation.Despite the above-described progress made in our understanding of ATs, their actual molecular mechanism of secretion remains uncertain. This is partially because the reported information is based on studies with different model AT proteins and nonhomogenous experimental approaches used by different laboratories, which sometimes produce data that are difficult to compare or may be conflicting. Here, we report a comparative study to determine conserved biochemical and functional properties found in AT C-terminal domains. Following a uniform experimental approach for six AT C-terminal domains selected from the gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria, we have investigated their capacities for the secretion of peptides and globular domains, their pore formation and oligomerization properties, and their requirement for an N-terminal α-helix for AT function and C-terminal domain stability. Our results shed light on the secretion mechanism of ATs from the conserved structural features found in their C-terminal domains. 相似文献
8.
Biophysical and Biochemical Heterogeneity of Purified Hepatitis B Antigen 总被引:14,自引:5,他引:9
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G. R. Dreesman F. B. Hollinger J. R. Suriano R. S. Fujioka J. P. Brunschwig J. L. Melnick 《Journal of virology》1972,10(3):469-476
Hepatitis B antigen of the D (a+, d+, y-) subtype was purified from plasma of apparently healthy persons and from hepatitis patients. The original samples contained 20- and 42-nm particles and tubular forms (20-nm diameter). Ultracentrifugation during the purification procedure yielded pellets which were then treated at pH 2.4. Both the large, 42-nm Dane particles and the tubular forms were lost during the acid treatment of the pelleted particles, yielding a preparation containing a mixture of particles approximately 20 and 25 nm in diameter. This difference in size was substantiated in that two distinct molecular weights were calculated from high-speed equilibrium data, 3.6 x 10(6) and 4.5 x 10(6). Further heterogeneity was observed in that hepatitis B antigenic activity was present in purified particles with an isoelectric pH of 4.0 and also in those with a pH of 4.4. No significant differences were observed in the gross amino acid composition of purified antigen obtained from plasma of three different persons. (125)I-labeled, purified antigen was found to contain six distinct polypeptides with molecular weights ranging from 10,000 to 39,000. 相似文献
9.
10.
The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly “transferable.” Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03∗) results in an unbiased energy function. We take as examples the 35-residue α-helical Villin HP35 and 37 residue β-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03∗ in explicit solvent fold to within 2.0 Å RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism. 相似文献
11.
Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization 总被引:2,自引:0,他引:2
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Tams Bakonyi Irmgard Derakhshifar Elvira Grabensteiner Norbert Nowotny 《Applied microbiology》2003,69(3):1504-1510
PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs. 相似文献
12.
Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering‐induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse‐grained and atomistic molecular dynamics simulations of two two‐domain constructs from the immunoglobulin‐like β‐sandwich fold. Each of these was experimentally shown to behave as the “sum of its parts,” that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two‐domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Proteins 2012; © 2012 Wiley Periodicals, Inc. 相似文献
13.
Biophysical and Biochemical Studies on Rhinovirus and Poliovirus II. Chemical and Hydrodynamic Analysis of the Rhinovirion 总被引:2,自引:2,他引:0
Chemical analysis of rhinovirus 14 revealed a ribonucleic acid (RNA) content of 29.8% and a high adenylic acid content (35%). A partial specific volume of 0.682 cm3/g was obtained for the rhinovirion. Rhinovirus and poliovirus had identical sedimentation coefficients of 158S. A diffusion coefficient of 1.71 × 10−7 cm2/sec was consistent with a hydrated diameter of 25 nm for the rhinovirion. The calculated molecular weights of the rhinovirion and its genome were 7.1 × 106 and 2.1 × 106 daltons, respectively. Sedimentation analysis of infectious RNA confirmed the similarity of the molecular size of the poliovirus and rhinovirus genomes. 相似文献
14.
In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling. Download video file.(105M, mov) 相似文献
15.
Esben M. Quistgaard Christian L?w Per Moberg Fatma Guettou Karthik Maddi P?r Nordlund 《PloS one》2013,8(8)
Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31. 相似文献
16.
Giorgio Lenaz Giovanna Curatola Laura Mazzanti Giovanna Parenti-Castelli 《Molecular and cellular biochemistry》1978,22(1):3-32
Summary The phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins.We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids.The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of -helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition.The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics.The conformational effect of anesthetics is not directly exerted on the porteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition. 相似文献
17.
《Journal of molecular biology》1996,264(2):377-389
CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphysor I). CI2 folds from a largely unstructured denatured state under physiological conditionsviaa transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys→3 ) is very similar for both proteins. 相似文献
18.
Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F-actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6-7 °C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 μM (W148R) and 0.56 μM (M202V), compared to the wild type ABD Kd of 7.0 μM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein. 相似文献
19.
Ruth L. Satter M. J. Morse Youngsook Lee Richard C. Crain Gary G. Cot Nava Moran 《Plant biology (Stuttgart, Germany)》1988,101(3):205-213
Leaflet movements in the legume Samanea saman are under joint control by light and a circadian oscillator. The movements are driven by massive fluxes of K+, Cl?, and H+ through pulvinar motor cell membranes. Light and the oscillator affect leaflet movements by altering the activity of ion transport systems. Some effects of light on ion transport may be mediated by the phosphatidylinositol (PI) cycle, since brief irradiation of the pulvinus with white light accelerates PI turnover. 相似文献
20.
Laura Smith Richard C. Page Zhen Xu Ekta Kohli Paul Litman Jay C. Nix Sujay S. Ithychanda Jianmin Liu Jun Qin Saurav Misra Carole M. Liedtke 《The Journal of biological chemistry》2010,285(22):17166-17176
Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells. 相似文献