Conformationally gated electron transfer studies of iso-1-cytochrome c

Protein folding is important because all proteins must fold to achieve their active conformer. In many cases, misfolding is the cause of disease and thus, understanding folding may lead to cures for disease. This thesis focuses on the dynamics and thermodynamics of partially unfolded states of proteins that lie near the bottom of a folding funnel. Various studies have provided evidence that partially unfolded proteins can be key intermediates in metabolic processes and in aggregation diseases. Partially unfolded proteins also provide insight into the types of structures that guide/misguide the process of protein folding. Less is known about how stability affects the roughness of an energy landscape at the bottom of the folding funnel. Histidine-heme alkaline conformers of cytochrome c are used as a model for a late folding intermediate or partially unfolded state that can be exploited to study roughness near the bottom of a folding funnel. In my thesis work, I have developed a novel method using electron transfer (ET) reactions to probe these conformational changes and thus provide insight into the dynamics of late folding processes not available through standard stopped-flow methods. This method has helped to probe two factors that affect dynamics near bottom of funnel: overall stability and the position of a residue in a loop that stabilizes a misfold. The main findings from my work include: a decrease in stability decreases the roughness of a folding funnel and changing the position of a residue responsible for misfolding strongly affects stability and dynamics of the misfolded state. The robustness pertaining to the gated ET method highlighted in this work is that it allows extraction of discrete rate constants for conformational changes under conditions where these rate constants cannot usually be measured directly. The method has also been applied to the dynamics of proline isomerization. The data demonstrate that rates of ET in proteins can be tuned efficiently using a combined strategy of modulating the sequence position and nature of the metal ligand involved in conformational gating. Thus, ET gates can be readily tuned for metabolic processes or the development of molecular switches

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Effect of an Ala81His Mutation on the Met80 Loop Dynamics of Iso-1-cytochrome <i>c</i>

An A81H variant of yeast iso-1-cytochrome <i>c</i> is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome <i>c</i>, 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 <i>c</i>. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome <i>c</i>. Δ<i>G</i>_u°(H₂O) is approximately 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome <i>c</i>, the native conformer is less stable than the His79-heme alkaline conformer. Δ<i>G</i>_u°(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 <i>c</i> arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, <i>k</i>_f,His81, relative to <i>k</i>_f,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 <i>c</i> for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes

Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity

Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity

The N- and C‑Terminal Domains Differentially Contribute to the Structure and Function of Dystrophin and Utrophin Tandem Calponin-Homology Domains

Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions

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 protein's 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