Structural effects of extracellular loop mutations in CFTR helical hairpins

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations – glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.     

Effects of mutations in apolipoprotein C-1 on the reconstitution and kinetic stability of discoidal lipoproteins

To probe the role of protein conformation in the formation and kinetic stability of discoidal lipoproteins, thermal unfolding and refolding studies were carried out using model lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and selected mutants of human apolipoprotein C-1 (apoC-1). Circular dichroism (CD) spectroscopy and electron microscopy show that the Q31P mutant, which has alpha-helical content in solution (33%) and on DMPC disks (67%) similar to that of the wild type (WT), forms disks of smaller diameter, = 13 nm, compared to 17 nm of the WT-DMPC disks. The L34P mutant, which is largely unfolded in solution, forms disks with alpha-helix content and diameter similar to those of the WT. The R23P mutant, which is fully unfolded in solution, forms disks that have similar diameter but reduced alpha-helix content (40%) compared to the WT-DMPC disks (65%). Remarkably, despite large variations in the alpha-helix content or the disk diameter among different mutant-DMPC complexes, the mutations have no significant effect on the unfolding rates or the Arrhenius activation energy of the disk denaturation, E(a) = 25-29 kcal/mol. This suggests that the kinetic stability of the discoidal complexes is dominated by the lipid-lipid rather than the protein-lipid interactions. In contrast to the heat denaturation, the lipoprotein reconstitution upon cooling monitored by CD and light scattering is significantly affected by mutations, with Q31P forming disks in the broadest and R23P in the narrowest temperature range. Our results suggest that the apolipoprotein helical structure in solution facilitates reconstitution of discoidal lipoproteins but has no significant effect on their kinetic stability.     

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.     

Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of unfolding of an integral membrane protein in mixed micelles

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl maltoside facilitate reversible and quantitative unfolding and refolding. The 4-transmembrane helix protein DsbB from the inner membrane of Escherichia coli unfolds in mixed micelles according to a three-state mechanism involving an unfolding intermediate I. The temperature dependence of the kinetics of this reaction between 15 degrees and 45 degrees C supports that unfolding from I to the denatured state D is accompanied by a significant decrease in heat capacity. For water-soluble proteins, the heat capacity increases upon unfolding, and this is generally interpreted as the increased binding of water to the protein as it unfolds, exposing more surface area. The decrease in DsbB's heat capacity upon unfolding is confirmed by independent thermal scans. The decrease in heat capacity is not an artifact of the use of mixed micelles, since the water soluble protein S6 shows conventional heat-capacity changes in detergent. We speculate that it reflects the binding of SDS to parts of DsbB that are solvent-exposed in the native DM-bound state. This implies that the periplasmic loops of DsbB are relatively unstructured. This anomalous thermodynamic behavior has not been observed for beta-barrel membrane proteins, probably because they do not bind SDS so extensively. Thus the thermodynamic behavior of membrane proteins appears to be intimately connected to their detergent-binding properties.     

Protein unfolding in detergents: effect of micelle structure,ionic strength,pH, and temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.     

Burst-phase expansion of native protein prior to global unfolding in SDS

Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein engineering the structure of an expanded but native-like intermediate that accumulates transiently in the unfolding reaction of the small protein S6 in the presence of SDS. The results show that the SDS micelles attack the native protein in the dead-time of the denaturation experiment, causing an expansion of the hydrophobic core prior to the major unfolding transition. We distinguish two forms of the unfolding intermediate that are correlated with the micellar structure. With spherical micelles, the expansion is seen mainly as a weakening of the interactions which anchor the two alpha-helices to the core of the S6 structure. With cylindrical micelles, prevalent at higher SDS concentrations, the expansion is more global and produces a species which closely resembles the transition-state structure for unfolding in GdmCl. Despite the highly weakened core, the micelle-associated intermediate displays cooperative unfolding, indicating a significant structural plasticity of the species on the native side of the folding barrier in the presence of SDS.     

Structural basis for unfolding pathway‐dependent stability of proteins: Vectorial unfolding versus global unfolding

Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig‐like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant‐induced “global unfolding” versus “vectorial unfolding” by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N‐terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through β‐sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N‐terminal structure probably through enhanced hydrophobic interactions.     

Biochemical and structural characterisation of cutinase mutants in the presence of the anionic surfactant AOT

The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.     

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS) and dodecyl maltoside (DM). This analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. Refolding and unfolding occur on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.     

An unfolding story of helical transmembrane proteins

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS-perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems, inverse micelles, and fragment association studies.     

Molecular dynamics simulation of the unfolding of individual bacteriorhodopsin helices in sodium dodecyl sulfate micelles

We report molecular dynamics simulations of the trends in the changes in secondary structure of the seven individual helices of bacteriorhodopsin when inserted into sodium dodecyl sulfate (SDS) micelles, and their dependence on the amino acid sequence. The results indicate that the partitioning of the helices in the micelles and their stability are dependent on the hydrophobicity of the transmembrane segments. Helices A, B, and E are stable and retain their initial secondary structure throughout the 100 ns simulation time. In contrast, helices C, D, F, and G show structural perturbations within the first 10 ns. The instabilities are localized near charged residues within the transmembrane segments. The overall structural instability of the helix is correlated with its partitioning to the surface of the micelle and its interaction with polar groups there. The in silico experiments were performed to complement the in vitro experiments that examined the partial denaturation of bacteriorhodopsin in SDS described in the preceding article (DOI 10.1021/bi201769z ). The simulations are consistent with the trends revealed by the experimental results but strongly underestimate the extent of helix to extended coil transformation. The reason may be either that the sampling time was not sufficiently long or, more interestingly, that interhelix residue interactions play a role in the unfolding of the helices.     

Effect of transmembrane helix packing on tryptophan and tyrosine environments in detergent-solubilized bacterio-opsin

Bacterio-opsin (bO) is folded in a nearly native conformation in mixed micelles of dimyristoyl phosphatidyl choline (DMPC) and 3-[(3-cholamidopropyl)-dimehtylamonio]-1-propane sulfonic acid (CHAPS), but bO is partially unfolded in sodium dodecyl sulfate (SDS). UV difference spectroscopy was used to study the changes in environment of bO aromatic amino acid side chains that occur upon partial unfolding. The UV difference spectra of peptides in CHAPS/DMPC minus peptides in SDS were measured for bO and the following subfragments of bO: C1 (residues 72–248), C2 (1–71), V1 (1–166), V2 (167–248), CB7 (119–145), CB9 (164–209), and CB10 (72–118). The spectra show that, in partially unfolded bO in SDS, the Tyr and Trp absorbance is blue-shifted. The difference spectra were compared to solvent perturbation difference spectra of N-acetyl-l-tyrosine ethyl ester and N-acetyl-l-tryptophanamide. The exposure change calculated from the difference spectra was found to correlate with the change in the number of van der Waals contacting atoms upon partial unfolding, and also with the number of transmembrane helical segments. This result suggests a simple experimental method of testing helix packing arrangements derived from hydropathy plots and model building.Abbreviations bO bacterio-opsin - bR bacteriorhodopsin - SDS sodium dodecyl sulfate - CHAPS 3-[(3-cholamidopropyl)-dimethylamonio]-1-propane sulfonic acid - C1 bacteriorhodopsin residues 72–248 - C2 1-71 - VI 1-166 - V2 167-248 - CB7 119-145 - CB9 164-209 - CB10 72-118 - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine - EDTA di-sodium ethylenediamine tetra-aceticacid     

Relative and regional stabilities of the hamster, mouse, rabbit, and bovine prion proteins toward urea unfolding assessed by nuclear magnetic resonance and circular dichroism spectroscopies

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808-19813] and for the bovine protein presented herein. The [urea](1/2) values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP(C) was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the β-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP(Sc) formation is linked with the global stability of PrP(C).     

Effects of magnesium sulfate on an unfolding step of human cyanomet myoglobin.

The effects of magnesium sulfate (MgSO4) on an unfolding step of human cyanomet myoglobin (Mb) were examined for wild-type and three L-->A mutant Mbs. The unfolding was induced at acidic pH (3.6-4.5) with various concentrations of MgSO4 (0-2 M). The monophasic process was monitored by visible absorption spectroscopy. We observed quite nonlinear delta G not equal to-[MgSO4] relations for all the Mbs. delta G not equal to-[MgCl2] relations were also determined for a comparative study. Thermodynamic evaluation of the results indicated that an upward reflection of delta G not equal to-[MgSO4] relations in high [MgSO4] is caused by the strong Hofmeister effect of the salt. Results obtained for three mutants (L29A, L72A, and L104A) at pH 4.0 and 4.5 were consistent with our previous observation that the structure of the transition state is determined by the stability of Mb cores in the balance with the pH conditions of unfolding (T. Konno and I. Morishima. 1993. Biochim. Biophys. Acta. 1162:93-98).     

How Ala-->Gly mutations in different helices affect the stability of the apomyoglobin molten globule

The apomyoglobin molten globule has a complex, partly folded structure with a folded A[B]GH subdomain; the factors determining its stability are not yet known in detail. Ala-->Gly mutations, made at solvent-exposed positions, are used to probe the role of helix propensity of individual helices in stabilizing the molten globule. Molten globule stability is measured by reversible urea unfolding, monitored both by circular dichroism and by tryptophan fluorescence. Two-state unfolding is tested by superposition of these two unfolding curves, and stability data are reported only for variants which satisfy the superposition test. Results for sites Q8 in the A helix and E109 in the G helix confirm that the helix propensities of the A and G helices both strongly affect molten globule stability, in contrast to results for the G65A/G73A double mutant which show that changing the helix propensity of the E-helix sequence has no significant stabilizing effect. Changing the helix propensity of the B-helix sequence with the G23A/G25A double mutant affects molten globule stability to an intermediate extent, confirming an earlier report that this mutant has increased stability. These results are consistent with the bipartite structure for the molten globule in which the A, G, and H helices are stably folded, while the long E helix is unfolded and the B helix has intermediate stability. Some differences are found in the shapes of the unfolding curves of different mutants even though they satisfy the superposition test for two-state unfolding, and possible explanations are discussed.     

An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

The extreme thermal stabilization achieved by the introduction of a disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 11152-11156] was attributed to the fixation of the loop region 56-69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfolding and autoproteolysis kinetics of pWT and G8C/N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS/PAGE. First-order rate constants (kobs) were evaluated by chevron plots (ln kobs vs. GdnHCl concentration). The kobs of unfolding showed a difference of nearly six orders of magnitude (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees C) between calcium saturation (at 100 mM CaCl2) and complete removal of calcium ions (in the presence of 100 mM EDTA). Analysis of the protease variant W55F indicated that calcium binding-site III, situated in the critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.     

Unfolding of beta-sheet proteins in SDS

Beta-sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two beta-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds around the critical micelle concentration. Isothermal titration calorimetry confirms that unfolding of TII27 sets in at lower SDS concentrations, although the total number of bound SDS molecules is similar at the end of unfolding. In mixed micelles with the nonionic detergent dodecyl maltoside, where the concentration of monomeric SDS is insignificant, the behavior of the two proteins converges. TII27 unfolds more slowly than TNfn3 in SDS and follows a two-mode behavior. Additionally TNfn3 shows inhibition of SDS unfolding at intermediate SDS concentrations. Mutagenic analysis suggests that the overall unfolding mechanism is similar to that observed in denaturant for both proteins. Our data confirm the kinetic robustness of beta-sheet proteins toward SDS. We suggest this is related to the inability of SDS to induce significant amounts of alpha-helix structure in these proteins as part of the denaturation process, forcing the protein to denature by global rather than local unfolding.