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1.
T Herning  K Yutani  Y Taniyama  M Kikuchi 《Biochemistry》1991,30(41):9882-9891
The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.  相似文献   

2.
When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.  相似文献   

3.
Cis proline mutants of ribonuclease A. I. Thermal stability.   总被引:8,自引:5,他引:3       下载免费PDF全文
A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.  相似文献   

4.
The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.  相似文献   

5.
Changes in protein stability can be achieved by making substitutions that increase or decrease the available conformations of the unfolded protein without altering the conformational freedom of the folded protein. Matthews and coworkers (1987) proposed that proline to alanine (P --> A) substitution would achieve this type of entropic destabilization. By comparing the Ramachandran area associated with alanine and proline residues, Matthews et al. estimated the unfolding entropy change resulting from P --> A substitution to be 4.8 cal mol(-1) K(-1). Although such an entropy difference would produce a substantial free energy change, accurately resolving such free energy changes into entropic and enthalpic components has been difficult. Here, we attempt to quantify the unfolding entropy change produced by P --> A substitution by amplifying the effect through multiple substitutions, and by decreasing the uncertainty in determining the unfolding entropy. Variants of a repeat protein, the Drosophila Notch ankyrin domain, were constructed with a varying number of P --> A substitutions at structurally conserved positions. Unfolding entropy values of the variants were determined from free energy measurements taken over a common temperature range using chemical denaturation. Our findings confirm the prediction that increasing the number of proline residues present in similar local environments increases the unfolding entropy. The average value of this increase in unfolding entropy is 7.7 +/- 4.2 cal mol(-1) K(-1), which is within error of the value estimated by Matthews et al. (1987).  相似文献   

6.
Effects of proline mutations on the folding of staphylococcal nuclease   总被引:5,自引:0,他引:5  
Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.  相似文献   

7.
Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures.  相似文献   

9.
Many secretory and several vacuolar proteins in higher plants contain hydroxylated proline residues. In many cases, hydroxyprolines in proteins are glycosylated with either arabinogalactan or oligoarabinose. We have previously shown that a sporamin precursor is O-glycosylated at the hydroxylated proline 36 residue with an arabinogalactan-type glycan when this protein is expressed in tobacco BY-2 cells (Matsuoka et al., 1995). Taking advantage of the fact that this is the only site of proline hydroxylation and glycosylation in sporamin, we analyzed the amino acid requirement for proline hydroxylation and arabinogalactosylation. We expressed several deletion constructs and many amino acid substitution mutants in tobacco cells and analyzed glycosylation and proline hydroxylation of the expressed sporamins. Hydroxylation of a proline residue requires the five amino acid sequence [AVSTG]-Pro-[AVSTGA]-[GAVPSTC]-[APS or acidic] (where Pro is the modification site) and glycosylation of hydroxyproline (Hyp) requires the seven amino acid sequence [not basic]-[not T]-[neither P, T, nor amide]-Hyp-[neither amide nor P]-[not amide]-[APST], although charged amino acids at the -2 position and basic amide residues at the +1 position relative to the modification site seem to inhibit the elongation of the arabinogalactan side chain. Based on the combination of these two requirements, we concluded that the sequence motif for efficient arabinogalactosylation, including the elongation of the glycan side chain, is [not basic]-[not T]-[AVSG]-Pro-[AVST]-[GAVPSTC]-[APS].  相似文献   

10.
It has long been understood that the proline residue has lower configurational entropy than any other amino acid residue due to pyrrolidine ring hindrance. The peptide bond between proline and its preceding amino acid (Xaa-Pro) typically exists as a mixture of cis- and trans-isomers in the unfolded protein. Cis–trans isomerization of Xaa-Pro peptide bonds are infrequent, but still occur in folded proteins. Therefore, the effects of the cis–trans isomerization equilibrium in both unfolded and folded states should be taken into account when estimating the stability contribution of a specific proline residue. In order to study the stability contribution of the four proline residues to the hyperthermophilic protein Ssh10b, in this work, we expressed and purified a series of Pro→Ala mutants of Ssh10b, and performed correlative unfolding experiments in detail. We proposed a new unfolding model including proline isomerization. The model predicts that the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between cis- and trans-isomers both in the unfolded and folded states, agreeing well with the experimental results.  相似文献   

11.
The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S-heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1-25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met-80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

12.
De Jesus M  Jin J  Guffanti AA  Krulwich TA 《Biochemistry》2005,44(38):12896-12904
Proline and glycine residues are well represented among functionally important residues in hydrophobic domains of membrane transport proteins, and several critical roles have been suggested for them. Here, the effects of mutational changes in membrane-embedded proline and glycine residues of Tet(L) were examined, with a focus on the conserved GP(155,156) dipeptide of motif C, a putative "antiporter motif". Mutation of Gly155 to cysteine resulted in a mutant Tet(L) that bound its tetracycline-divalent metal (Tc-Me2+) substrate but did not catalyze efflux or exchange of Tc-Me2+ or catalyze uptake or exchange of Rb+ which was used to monitor the coupling ion. These results support suggestions that this region is involved in the conformational changes required for translocation. Mutations in Pro156 resulted in reduction (P156G) or loss (P156A or P156C) of Tc-Me2+ efflux capacity. All three Pro156 mutants exhibited a K+ leak (monitored by 86Rb+ fluxes) that was not observed in wild-type Tet(L). A similar leak was observed in a mutant in a membrane-embedded proline residue elsewhere in the Tet(L) protein (P175C) as well as in a P156C mutant of related antiporter Tet(K). These findings are consistent with roles proposed for membrane-embedded prolines in tight helix packing. Patterns of Tc resistance conferred by additional Tet(L) mutants indicate important roles for another GP dipeptide in transmembrane segment (TMS) X as well as for membrane-embedded glycine residues in TMS XIII.  相似文献   

13.
To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.  相似文献   

15.
Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.  相似文献   

16.
Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.  相似文献   

17.
5-HT3 receptors possess a number of highly conserved proline residues. We changed each of these to alanine, expressed the mutants as homomeric 5-HT3A receptors in HEK293 cells, and analyzed them with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Pro56, Pro104, Pro123, and Pro170 resulted in ablation of radioligand binding, whereas mutation of Pro257 and Pro301 did not. Only the latter were expressed at the plasma membrane but were non-functional. Thus the former, which are in the N-terminal domain, may be involved in forming correct receptor structure, while those in the transmembrane region (Pro257 and Pro301) are necessary for the function of the protein. To explore the conformational preference (propensity) of these residues we examined the proportion of cis-prolines and the influence of adjacent residues in known protein structures. 4.7% of prolines in the protein data base were in the cis conformation, and the distribution of amino acids adjacent to cis-prolines was not randomly distributed. Comparison of the proportion of each amino acid residue adjacent to a cis-proline revealed that aromatic and bend-facilitating residues were favored while those with beta-branched chains were not. Thus five residues (Gly, Pro, Tyr, Trp, Phe) and three residues (Pro, Tyr, Phe) were found more frequently than expected before and after cis-prolines respectively, whereas five residues (Val, Ile, Leu, Asp, Thr) and two residues (Asp, Glu) were found less frequently. Of the 20 proline residues in the 5-HT3A receptor subunit only Pro170 has adjacent residues that are favorable. Mutating these to non-favorable residues resulted in ablation of ligand binding, whereas replacement with alternative favorable residues did not. We therefore propose that Pro170, which is part of the characteristic cys-loop found in this family of proteins, may be in the cis conformation.  相似文献   

18.
Site-directed mutagenesis has frequently been used to replace proline with other amino acids in order to determine if proline isomerization is responsible for a slow phase during refolding. Replacement of Pro 85 with alanine in cellular retinoic acid binding protein I (CRABP-I) abolished the slowest refolding phase, suggesting that this phase is due to proline isomerization in the unfolded state. To further test this assumption, we mutated Pro 85 to valine, which is the conservative replacement in the two most closely related proteins in the family (cellular retinoic acid binding protein II and cellular retinol binding protein I). The mutant protein was about 1 kcal/mole more stable than wild type. Retinoic acid bound equally well to wild type and P85V-CRABP I, confirming the functional integrity of this mutation. The refolding and unfolding kinetics of the wild-type and mutant proteins were characterized by stopped flow fluorescence and circular dichroism. The mutant P85V protein refolded with three kinetic transitions, the same number as wild-type protein. This result conflicts with the P85A mutant, which lost the slowest refolding rate. The P85V mutation also lacked a kinetic unfolding intermediate found for wild-type protein. These data suggest that proline isomerization may not be responsible for the slowest folding phase of CRABP I. As such, the loss of a slow refolding phase upon mutation of a proline residue may not be diagnostic for proline isomerization effects on protein folding.  相似文献   

19.
Kuwayama S  Imai H  Hirano T  Terakita A  Shichida Y 《Biochemistry》2002,41(51):15245-15252
To identify the amino acid residue(s) responsible for the difference in the molecular properties between rod and cone pigments, we have prepared chicken green mutants where each of the residues (Val77, Gly144, and Pro189) completely conserved in the cone pigments was replaced with the residue in the rod pigment rhodopsin. Among the mutants, the P189I mutant showed an expression level in cultured HEK293 cells and a thermal stability higher than did the wild-type chicken green. The mutation caused a reduced decay rate of the meta II intermediate, while the mutation of the wild-type chicken rhodopsin at position 189 (I189P) resulted in an increased decay rate. The additional mutation at position 122, the previously reported site where the amino acid residue is one of the determinants of the meta II decay rate, converted the meta II decay rate into that observed in the wild-type chicken rhodopsin. These results suggest that the difference in the meta II decay rate between the chicken green and rhodopsin is due to the difference in the amino acid residues at positions 189 and 122. The completely conserved nature of proline at position 189 could provide a clue to the molecular evolution of the pigments.  相似文献   

20.
Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.  相似文献   

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