The folding kinetics and thermodynamics of the Fyn-SH3 domain

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.     

Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability

The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature-denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: comparison with the intact protein

Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1-56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N-terminal domain are very similar to those previously measured for the corresponding region in the full-length protein (Lillemoen J et al., 1997, J Mol Biol 268:482-493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N-terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.     

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Mutational analysis of the BPTI folding pathway: II. Effects of aromatic-->leucine substitutions on folding kinetics and thermodynamics

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.     

Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54 degrees C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a delta GH2O of 3.1 kcal mol-1 at pH 8.0 and 25 degrees C. The chemical denaturation is broad with an m-value of 760 cal mol-1 M-1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31 degrees C. Chemical denaturations at 4 degrees C give an m-value of 740 cal mol-1 M-1, similar to the wild-type, but the stability delta GH2O is only 1.4 kcal mol-1. Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.     

Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain

We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y. -S. Chang, and W. Chang, J. Virol. 72:1577-1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.     

Conformational stability of muscle acylphosphatase: the role of temperature, denaturant concentration, and pH

The conformational stability (delta G) of muscle acylphosphatase, a small alpha/beta globular protein, has been determined as a function of temperature, urea concentration, and pH. A combination of thermally induced and urea-induced unfolding, monitored by far-UV circular dichroism, was used to define the conformational stability over a wide range of temperature. Through analysis of all these data, the heat capacity change upon unfolding (delta Cp) could be estimated, allowing the determination of the temperature dependence of the main thermodynamic functions (delta G, delta H, delta S). Thermal unfolding in the presence of urea made it possible to extend such thermodynamic analysis to examine these parameters as a function of urea concentration. The results indicate that acylphosphatase is a relatively unstable protein with a delta G(H2O) of 22 +/- 1 kJ mol-1 at pH 7 and 25 degrees C. The midpoints of both thermal and chemical denaturation are also relatively low. Urea denaturation curves over the pH range 2-12 have allowed the pH dependence of delta G to be determined and indicate that the maximum stability of the protein occurs near pH 5.5. While the dependence of delta G on urea (the m value) does not vary with temperature, a significant increase has been found at low pH values, suggesting that the overall dimensions of the unfolded state are significantly affected by the number of charges within the polypeptide chain. The comparison of these data with those from other small proteins indicates that the pattern of conformational stability is defined by individual sequences and not by the overall structural fold.     

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (beta-TPA). Within 10 min, beta-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens.     

Fragment reconstitution of a small protein: folding energetics of the reconstituted immunoglobulin binding domain B1 of streptococcal protein G

To elucidate early stages in protein folding, we have adopted a fragment reconstitution method for small proteins. This approach is expected to provide nuclei for protein folding and to allow us to investigate folding mechanisms. In previous work [Kobayashi, N., et al. (1995) FEBS Lett. 366, 99-103.] we demonstrated the association of two complementary fragments, derived from the immunoglobulin G-binding domain B1 of streptococcal Protein G, and showed the structural similarity between the reconstituted domain and the uncleaved wild-type domain. In this work we have further characterized the reconstituted domain as well as the uncleaved domain thermodynamically by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. Although composed of short peptide fragments not linked by covalent bonds, the reconstituted domain showed a typical folding/unfolding curve in both DSC and CD melting measurements and behaved like a globular protein. The domain was not very stable, and the small value of the Gibbs free energy corresponded to the class of the weakest protein-protein binding systems. The denaturation temperature of 0. 78 mM solution was 313 K at pH 5.9 as measured by DSC, which was more than 40 degrees lower than the uncleaved domain. This apparent instability was primarily caused by entropic disadvantage attributed to a bimolecular reaction. The temperature dependence of the enthalpy change from the folded to the unfolded state was almost identical for the reconstituted domain and the uncleaved one. This indicates that most of the noncovalent intramolecular interactions stabilizing the native structure, such as hydrogen bonding and hydrophobic interactions, are regenerated in the reconstituted domain. By comparing the equilibrium constants of the reconstituted and uncleaved domains, we determined the effective concentration to be approximately 6 M at 298 K. Structure-based estimation of the thermodynamic properties from the values of accessible surface areas showed that approximately 35% of the total heat capacity change and approximately 25% of the total enthalpy change can be attributed to the interchain interaction at 298 K. Furthermore, the folding/unfolding equilibrium of beta-hairpin structure of the fragment 41-56 alone was also characterized. These analyses allow us to envision the microdomain folding mechanism of the Protein G B1 domain, in which segment 41-56 first forms a stable beta-hairpin structure and then collides with segment 1-40, followed by spontaneous folding of the whole molecule.     

Local interactions and the optimization of protein folding

Haemodynamic effects of hypertonic saline solutions (HSS) have been extensively studied in animals and humans. Hypertonic sodium chloride (7.5%, 2,500 mOsm.L-1) either alone or combined with colloids, remains the standard solution. The haemodynamic response of HSS observed during treatment of hypovolaemic shock is explained by 1) an increase in preload due to the expansion of the plasma volume and a musculocutaneous vasoconstriction and 2) a decrease in systemic vascular resistance and afterload. A myocardial stimulation has been shown in various experimental conditions and in humans. However, the clinical relevance of this inotropic effect is questionable. Haemorrhagic shock is the main indication for small volume resuscitation with HSS. Other potential situations for the use of HSS are volume replacement in perioperative period, septic shock or burn injury and cardiopulmonary resuscitation. Before recommending the clinical use of HSS, additional clinical studies are required to substantiate the benefits of HSS over colloids.     

The function of the p190 Rho GTPase-activating protein is controlled by its N-terminal GTP binding domain

p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser36 --> Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was inhibited by wild type HA-p190, but JNK activity was enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibited the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain.     

The conserved carboxyl terminus and zinc finger-like domain of the co-chaperone Ydj1 assist Hsp70 in protein folding

Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179-384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179-384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell.     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.