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1.
Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic stability of low abundant proteins in cell lysates. Previously, it was demonstrated that protein stability can be determined quantitatively by measuring the fraction of folded proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show that thermodynamic stability of low abundant proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western). To demonstrate the reliability of this method, we determined the thermodynamic stability of recombinant human H‐ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic stability determined with pure H‐ras revealed that Pulse and Western is a reliable way to monitor protein stability in cell lysates and the stability of H‐ras is not affected by other proteins present in cell lysates. This method allows the investigation of conformational energetics of proteins in cell lysates without cloning, purification, or labeling.  相似文献   

2.
Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (Kd) of the complex from unfolding kinetic constants, which is also in good agreement with known Kd values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.  相似文献   

3.
Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A560). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (Cm) shows excellent agreement with that determined by A560. The Cm values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A560. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.  相似文献   

4.
Transient partial unfolding of proteins under native conditions may have significant consequences in the biochemical and biophysical properties of proteins. Native-state proteolysis offers a facile way to investigate the thermodynamic and kinetic accessibilities of partially unfolded forms (cleavable forms) under native conditions. However, determination of the structure of the cleavable form, which is populated only transiently, remains challenging. Although in some cases partially cleaved products from proteolysis provide information on the structure of this elusive form, proteolysis of many proteins does not accumulate detectable intermediates. Here, we describe a systematic approach to determining structures of cleavable forms by protein engineering and native-state proteolysis. By devising φc analysis, which is analogous to conventional φ analysis, we have determined the structure of the cleavable form of Escherichia coli maltose-binding protein (MBP), which does not accumulate any partially cleaved products. We mutated 10 buried residues in MBP to alanine and determined φc values from the effects of the mutations on global stability and proteolytic susceptibility. The result of this analysis suggests that two C-terminal helices in MBP are unfolded in their cleavable form. The effect of ligand binding on proteolytic susceptibility and C-terminal deletion mutations also confirms the proposed structure. Our approach and methodology are generally applicable not only in elucidating the mechanism of proteolysis but also in investigating other important processes involving partial unfolding under native conditions such as protein misfolding and aggregation.  相似文献   

5.
Selection of stably folded proteins by phage-display with proteolysis.   总被引:3,自引:0,他引:3  
To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.  相似文献   

6.
HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high stability with a little increase of secondary structure percentage as compared with the full-length protein. We anticipated that the N-terminally truncated protein possesses a more compact structure. By sequence analysis, the proteolytic fragment shares a great similarity with DUF589 domain, a previously identified domain with unknown function. This novel domain is highly conserved in Thy28 proteins and is worthy of structural and functional studies. We have subcloned this homologous domain from HSPC144 protein and purified to homogeneity for structure analysis. The (15)N and (15)N/(13)C-labeled DUF589 domain samples have been prepared successfully and determination of the NMR structure is in progress.  相似文献   

7.
The chromosomal YoeB-YefM toxin-antitoxin module common to numerous strains of bacteria is presumed to have a significant role in survival under stringent conditions. Recently we showed that the purified YefM antitoxin is a natively unfolded protein, as we previously reported for the Phd antitoxin in the P1 phage Doc-Phd toxin-antitoxin system. Here we report the purification and structural properties of the YoeB toxin and present physical evidence for the existence of a tight YoeB.YefM polypeptide complex in solution. YoeB and YefM proteins co-eluted as single peaks in sequential Ni-affinity FPLC and Q-Sepharose ion-exchange chromatography implying the formation of a YoeB.YefM complex. The unstable antitoxin was removed from the mixture by natural proteolysis, and the residual YoeB protein was purified using ion exchange chromatography. Fluorescence anisotropy studies of the purified YoeB and YefM proteins showed a 2:1 stoichiometry of the complex, providing direct evidence for a physical complex between the proteins. Near- and far-UV circular dichroism spectroscopy of the purified toxin revealed that, similar to the Doc toxin, YoeB is a well-folded protein. Thermal denaturation experiments confirmed the conformational stability of the YoeB toxin, which underwent reversible thermal unfolding at temperatures up to 56 degrees C. The thermodynamic features of the toxin-antitoxin complex were similar. Taken together, our results support the notion of a correlation between differential physiological and structural stability in toxin-antitoxin modules.  相似文献   

8.
The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.  相似文献   

9.
The synchrotron X-ray protein radiolysis technique is based on a quantitative determination of the extent and the site of millisecond radiolytic oxidation of amino-acid side chains by mass spectrometry. The amino acids most susceptible to radiolytic oxidation are cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, and leucine. These residues serve as reactive markers within a protein structure that can be used to monitor changes in solvent accessibility during folding or as part of macromolecular interactions. To monitor the unfolding, the extent of radiolytic products of side chains of reactive amino acids is quantitatively measured by mass spectrometry as a function of the denaturant concentration following proteolysis. This approach provides site-specific unfolding isotherms for various segments of a protein without the use of mutation or labeling techniques. Application of this technique to the equilibrium urea unfolding of apomyoglobin at pH 7.8 has demonstrated the cooperative unfolding of helices A to C consistent with midpoints, DeltaG, and m values derived from fluorescence data. The G helix, in contrast, showed a local unfolding behavior. The similarity of the thermodynamic data derived by this synchrotron-based method for helix A (containing two oxidizable tryptophan residues) to that of the fluorescence data indicates that the limited oxidation of proteins by exposure to X-rays on millisecond timescales does not alter the structure of apomyglobin. This supports the viability of the method for the study of protein folding and the mapping of protein interaction sites.  相似文献   

10.
Efficient methods for quantifying dissociation constants have become increasingly important for high‐throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein–ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein–protein complex involving the β‐lactamase TEM‐1 and various β‐lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, Cm, of TEM‐1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein–protein complexes. From a small set (n = 4) of TEM‐1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔCm was observed. From this “calibration curve,” accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis‐derived ΔCm values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high‐throughput mutagenesis binding studies.  相似文献   

11.
Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D2O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D2O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.  相似文献   

12.
To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as “one-pot analysis” which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.  相似文献   

13.

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

  相似文献   

14.
This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.  相似文献   

15.
H Iwai  A Plückthun 《FEBS letters》1999,459(2):166-172
We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.  相似文献   

16.
17.
Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P96. Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.  相似文献   

18.
19.
We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.  相似文献   

20.
We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.  相似文献   

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