Tural Aksel

Size and Sequence and the Volume Change of Protein Folding

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chateliers principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the proteins conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

The contribution of entropy, enthalpy, and hydrophobic desolvation to cooperativity in repeat-protein folding

Cooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity.

Ensemble Force Changes that Result from Human Cardiac Myosin Mutations and a Small-Molecule Effector

SUMMARY Cardiomyopathies due to mutations in human β-cardiac myosin are a significant cause of heart failure, sudden death, and arrhythmia. To understand the underlying molecular basis of changes in the contractile system’s force production due to such mutations and search for potential drugs that restore force generation, an in vitro assay is necessary to evaluate cardiac myosin’s ensemble force using purified proteins. Here, we characterize the ensemble force of human α- and β-cardiac myosin isoforms and those of β-cardiac myosins carrying left ventricular non-compaction (M531R) and dilated cardiomyopathy (S532P) mutations using a utrophin-based loaded in vitro motility assay and new filament-tracking software. Our results show that human α- and β-cardiac myosin, as well as the mutants, show opposite mechanical and enzymatic phenotypes with respect to each other. We also show that omecamtiv mecarbil, a previously discovered cardiac-specific myosin activator, increases β-cardiac myosin force generation.

Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

ABSTRACT Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. Summary: The underlying molecular basis of genetic-based cardiomyopathy diseases is largely unknown. This review describes recent molecular studies that have used human cardiac proteins to begin to elucidate the mechanisms whereby mutations cause disease.

Direct NMR detection of bifurcated hydrogen bonding in the α-helix N-caps of ankyrin repeat proteins.

In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the Nδ1 atom of histidine and both the backbone N–H and side-chain Oγ-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of 2hJNN HBCs supports that Thr N–H···Nδ1 His H-bonds within internal repeats are stronger (∼4 Hz) than in the solvent exposed C-terminal AR (∼2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant 2hJ and 1hJ couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in 2hJNN, attributed to optimization of the Val N–H···Nδ1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.