共查询到20条相似文献,搜索用时 15 毫秒
1.
T Sivaraman T K Kumar Y T Tu W Wang W Y Lin H M Chen C Yu 《Biochemical and biophysical research communications》1999,260(1):284-288
The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding. 相似文献
2.
Ebersbach H Fiedler E Scheuermann T Fiedler M Stubbs MT Reimann C Proetzel G Rudolph R Fiedler U 《Journal of molecular biology》2007,372(1):172-185
The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules. 相似文献
3.
Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble. 相似文献
4.
Samuel D Kumar TK Ganesh G Jayaraman G Yang PW Chang MM Trivedi VD Wang SL Hwang KC Chang DK Yu C 《Protein science : a publication of the Protein Society》2000,9(2):344-352
The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone. 相似文献
5.
The refolding kinetics of Cobrotoxin (CBTX), a small all beta-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen-deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all beta-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates. 相似文献
6.
Chow MK Amin AA Fulton KF Whisstock JC Buckle AM Bottomley SP 《Protein expression and purification》2006,46(1):166-171
The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources--REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community. 相似文献
7.
High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form. 相似文献
8.
While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason. 相似文献
9.
A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation. 相似文献
10.
Here, we present a systematic analysis of the open-faced beta-sheet topologies in a set of non-redundant protein domain structures; in particular, we focus on the topological diversity of four-stranded beta-sheet motifs. Of the 96 topologies that are possible for a four-stranded beta-sheet, 42 were identified in known protein structures. Of these, four account for 50% of the structures that we have studied. Two sets of the topologies that were not observed may represent the section of the topological space that is not readily accessible to proteins on either thermodynamic or kinetic grounds. The first set contains topologies with alternating parallel and antiparallel beta-ladders. Their rare occurrence reflects the expectation that it is energetically unfavorable to match different hydrogen bonding patterns. The polypeptide chains in the second set of topologies go through convoluted paths and are expected to experience great kinetic frustrations during the folding processes. A knowledge of the potential causes for the topological preference of small beta-sheets also helps us to understand the topological properties of larger beta-sheet structures which frequently contain four-stranded motifs. The notion that protein topologies can only be taken from a confined and discrete space has important implications for structural genomics. 相似文献
11.
Wong BS Vénien-Bryan C Williamson RA Burton DR Gambetti P Sy MS Brown DR Jones IM 《Biochemical and biophysical research communications》2000,276(3):1217-1224
We have shown previously that normal mouse prion protein (MoPrP) binds copper ions during protein refolding and acquires antioxidant activity. In this report, we probe the structure of the copper refolded form of MoPrP to determine how copper binding alters the secondary and tertiary features of the protein. Circular dichroism showed that recombinant MoPrP prepared in the presence of copper (as Cu(++)) showed an increased signal in the 210-220 nm range of the spectrum. Changes in protein conformation were localised to the N-terminal region of MoPrP using a panel of antibodies to assess epitope accessibility. The copper refolded recombinant prion protein had reduced proteinase K (PK) sensitivity when compared to the non-copper liganded form. Reduced PK sensitivity was not due to aggregation however as high resolution electron microscopy showed a homogenous preparation with little aggregate when compared to the non-copper form. Finally, disruption of the single disulphide linkage in MoPrP significantly diminished the antioxidant activity of the copper refolded form suggesting that activity was not solely dependent on bound copper but also on a conformation enabled by the formation of the disulphide bond. 相似文献
12.
Ader N Brindley MA Avila M Origgi FC Langedijk JP Örvell C Vandevelde M Zurbriggen A Plemper RK Plattet P 《The Journal of biological chemistry》2012,287(20):16324-16334
It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism. 相似文献
13.
A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding
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Reader JS Van Nuland NA Thompson GS Ferguson SJ Dobson CM Radford SE 《Protein science : a publication of the Protein Society》2001,10(6):1216-1224
The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein. 相似文献
14.
We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100–140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3–4 s; and 3), 60–130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore. 相似文献
15.
Jones S Reader JS Healy M Capaldi AP Ashcroft AE Kalverda AP Smith DA Radford SE 《Biochemistry》2000,39(19):5672-5682
Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold. 相似文献
16.
The expression of heterologous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. To fully exploit the production capacity of the cells, efficient strategies for further processing have to be developed. While in lab scale matrix assisted refolding techniques, especially of histidine-tagged proteins have become very popular, in production scale refolding by dilution is still predominant due to its simplicity. However scaling up dilution processes leads to large volumes and low protein concentration. This is a heavy burden both for liquid handling and for subsequent downstream processing steps. Process development aims to operate at uniform, reproducible conditions, to reduce costs to a minimum and to guarantee the required quality of the product. The general refolding kinetics, exploration of appropriate refolding conditions are reviewed. The major refolding operations such as dilution, matrix assisted refolding, pressure driven refolding or continuous refolding applications are discussed in view of industrial applicability. 相似文献
17.
Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology. 相似文献
18.
Brockwell DJ Paci E Zinober RC Beddard GS Olmsted PD Smith DA Perham RN Radford SE 《Nature structural biology》2003,10(9):731-737
Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension. 相似文献
19.
The formation of beta-sheet domains in proteins involves five energetically important factors: the formation of networks of hydrogen bonds and hydrophobic faces, and the residue propensities, or preferences, to be found at the edges of the beta-sheet, to adopt the extended conformation, and to make contact with other residues. These relative energy contributions define a potential energy function. Here, we show how optimizing this potential energy function reveals the formation of hydrophobic faces as the utmost factor. The potential energy function was optimized to minimize the Z-scores of the native topologies among the exhaustive sets of over 400 different beta-sheets. These results corroborate with experimental data that showed the environment of a protein is an important modulator of beta-sheet folding. The contact propensities were found to be the least important, which could explain the poor predictive power of beta-strand alignment methods based on pair-wise contact matrices. 相似文献
20.
Nakagawa T Shimizu H Link K Koide A Koide S Tamura A 《Journal of molecular biology》2002,323(4):751-762
Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition. 相似文献