Swati Bandi

Probing the bottom of a folding funnel using conformationally gated electron transfer reactions.

The effect of global stability on the kinetics of interconversion between the native (N) and a compact, partially unfolded form (I) of iso-1-cytochrome c stabilized by His73-heme ligation is investigated using a novel conformationally gated ET method. For the K73H variant and the 2-fold less stable AcH73 variant, the N and I conformers are of nearly equal stability at pH 7.5. The pH jump kinetic data yield kobs = kNI + kIN of 35-40 s-1 at final pH values from 6 to 8 for the AcH73 variant, about 3-fold faster than for the more stable K73H variant. Gated ET measurements give kNI = 28 s-1 and kIN = 13 s-1 for the AcH73 variant, 10- and 2-fold greater than that for the more stable K73H variant. Thus, funneled landscapes have evolved such that loss of global stability lowers barriers at the bottom of a folding funnel, still allowing for efficient folding.

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods.

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin †

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N‐terminal actin‐binding domain (N‐ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N‐ABD and compared with that of dystrophin. Our results show that utrophin N‐ABD has spectroscopic properties similar to dystrophin N‐ABD. However, utrophin N‐ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half‐life compared to dystrophin. In addition, utrophin N‐ABD aggregates to a lesser extent compared with dystrophin N‐ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N‐ABD mutations analogous in position to the dystrophin disease‐causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross‐β aggregates, indicating a possible role for utrophin mutations in disease mechanisms. Proteins 2012;

Effect of an Ala81His mutation on the Met80 loop dynamics of iso-1-cytochrome c.

An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric size and to provide a probe of the dynamics of the heme crevice through measurement of the thermodynamics and kinetics of the His81-mediated alkaline conformational transition of A81H iso-1-cytochrome c. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome c. ΔGu°(H2O) is approximately 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome c, the native conformer is less stable than the His79-heme alkaline conformer. ΔGu°(H2O) is approximately -0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonstrate that this stabilization of the native conformer in A81H iso-1-cytochrome c arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, kf,His81, relative to kf,His79 for formation of the His79-heme alkaline conformer, which forms by a mechanism similar to that observed for the His81-heme alkaline conformer. The result is discussed in terms of the effect of global protein stability on protein dynamics and in terms of optimization of the sequence of cytochrome c for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes.

Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the proteins overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

The actin binding affinity of the utrophin tandem calponin-homology domain is primarily determined by its N-terminal domain.

The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.

The C-terminal domain of the utrophin tandem calponin-homology domain appears to be thermodynamically and kinetically more stable than the full-length protein.

Domains are in general less stable than the corresponding full-length proteins. Human utrophin tandem calponin-homology (CH) domain seems to be an exception. Reversible, equilibrium denaturant melts indicate that the isolated C-terminal domain (CH2) is thermodynamically more stable than the tandem CH domain. Thermal melts show that CH2 unfolds at a temperature higher than that at which the full-length protein unfolds. Stopped-flow kinetics indicates that CH2 unfolds slower than the full-length protein, indicating its higher kinetic stability. Thus, the utrophin tandem CH domain may be one of the few proteins in which an isolated domain is more stable than the corresponding full-length protein.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy

We examined how polysorbate 20 (PS20; Tween 20) and polysorbate 80 (PS80; Tween 80) affect the higher-order structure of a monoclonal antibody (mAb) and its antigen-binding (Fab) and crystallizable (Fc) fragments, using near-UV circular dichroism and 2D nuclear magnetic resonance (NMR). Both polysorbates bind to the mAb with submillimolar affinity. Binding causes significant changes in the tertiary structure of mAb with no changes in its secondary structure. 2D 13C-1H methyl NMR indicates that with increasing concentration of polysorbates, the Fab region showed a decrease in crosspeak volumes. In addition to volume changes, PS20 caused significant changes in the chemical shifts compared to no changes in the case of PS80. No such changes in crosspeak volumes or chemical shifts were observed in the case of Fc region, indicating that polysorbates predominantly affect the Fab region compared to the Fc region. This differential effect of polysorbates on the Fab and Fc regions was because of the lesser thermodynamic stability of the Fab compared to the Fc. These results further indicate that PS80 is the preferred polysorbate for this mAb formulation, because it offers higher protection against aggregation, causes lesser structural perturbation, and has weaker binding affinity with fewer binding sites compared to PS20.

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70–85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild-type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild-type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer.

A cytochrome C electron transfer switch modulated by heme ligation and isomerization of a peptidyl‐prolyl bond

Intermolecular electron transfer (ET) between hexaamineruthenium(II), a(6) Ru(2+) , and a K73H/K79A variant of iso-1-cytochrome c, iso-1-Cytc, is used to study conformational ET switches mediated by His73-heme ligation and cis to trans isomerization of the Ile75-Pro76 peptidyl-prolyl bond of iso-1-Cytc. The biomolecular rate constant for ET to the native state of K73H/K79A iso-1-Cytc is ∼270 mM(-1) s(-1) near neutral pH. The unimolecular conformational ET switches due to His73-heme ligation and the Ile75-Pro76 peptidyl-prolyl bond gate ET at rate constants of 5 to 10 s(-1) and 0.05 to 0.06 s(-1) . Thus, at 1 mM a(6) Ru(2+) , these conformational ET switches slow electron transfer by about 50- and 5000-fold, respectively. The conformational ET switches are populated between pH 5 and 7, providing a means of modulating ET in this redox protein over several orders of magnitude by simply changing pH. The conformationally-gated ET measurements are analyzed in the context of previous pH jump measurements on the His73-heme alkaline transition of K73H/K79A iso-1-Cytc. The ability to obtain microscopic rate constants with conformationally-gated ET measurements has allowed more precise determination of the pK(a) s of the three ionizable groups that mediate population of the His73-heme ET switch. We have also been able to show that the ionizable group with a pK(a) near 9 stabilizes the His73-heme conformer relative to the native state of iso-1-Cytc and that contrary to the conclusions from our pH jump studies, this ionization does not strongly affect the rate of the Ile75-Pro76 peptidyl-prolyl isomerization.