Kinetics and Energetics of Fundamental Events in Peptide and Protein (Un)Folding

Abstract

One of the major challenges in the field of biophysical chemistry is the study of the mechanisms of protein folding, i.e., how an unstructured polypeptide chain can rapidly adopt an unique, densely packed, three-dimensional structure. Modifications in the folding kinetics and transitions to “misfolded” states are thought to be involved in the pathogenesis of many diseases commonly known as conformational disorders, including cystic fibrosis, type II diabetes, Alzheimer’s and Parkinson’s diseases. The global folding of proteins typically occurs in the millisecond to second time scale, but the underlying fundamental molecular events such as beta-hairpin or alpha-helix folding occur in the microsecond or faster time scale. The methods generally employed for studying folding or unfolding kinetics are typically limited to the time range of milliseconds or slower because the process usually involves mixing buffers in stopped-flow apparatus. A new generation of kinetic experiments has emerged to investigate the mechanisms of protein folding on the previously inaccessible sub-millisecond time scale. As a result, the earliest conformational events related to folding, occurring within microseconds or less may now be measured experimentally and interpreted. Laser-triggered fast initiation of the folding/unfolding reaction coupled with fast detection techniques provides the tools to probe these events in detail.

As the native conformations of peptides and proteins are normally sensitive to pH, conformational changes can be initiated by a pH change. Here, we propose to use a laser-triggered pH change, which provides an interesting way to probe the early events in protein folding/unfolding. With this technique, the pH jump occurs in few a nanoseconds and lasts for several milliseconds. This fast proton gradient protonates/deprotonates ionizable residues of a protein or peptide producing different charged species, and consequently conformational changes. Photothermal methods, like time-resolved photoacoustic calorimetry (TR-PAC) have the ability to measure accurately enthalpy and molar volume changes for reactions occurring with lifetimes in the nanoseconds to tens of microsecond time range. TR-PAC is likely the technique of choice to study fast events in protein folding when a spectroscopic technique is not applicable either because the intermediates are “dark” or too short lived. In this work, we propose to use a pH-jump methodology to trigger the folding/unfolding events, coupled with TR-PAC to monitor the energetics and kinetics of pH-dependent folding/unfolding of peptides and proteins.

Throughout this thesis, simple molecules such as amino acid models, an alpha-helical peptide and proteins were subjects of study to investigate protein folding, using a combination of pH-jump and TR-PAC detection, and fluorescence spectroscopy. Complementary equilibrium structural information was obtained by methodologies such as nuclear magnetic resonance (NMR) and circular dichroism (CD). In the first stage, the association of pH-jump and TR-PAC detection was used to investigate the protonation of amino acids models, which corresponds to the earliest step in protein or peptide pH-induced folding/unfolding events. On a second stage, a small peptide similar to the C-Peptide of RNase A, forming a stable alpha-helix in aqueous solution, was selected as a model system to explore the energetics and dynamics of alpha-helix formation. The C-Peptide analogue investigated was RN80 synthesized with specific side-chain interactions, namely a salt-bridge and a pi-stacking interaction that contributes to the alpha-helix stability. Finally, we propose the application of these methods to the study of proteins, searching for intermediate states during their folding/unfolding processes, such as molten globule states. As a protein system model, bovine serum albumin (BSA) has been selected because it undergoes partial unfolding transitions under acid conditions.

In a parallel effort, the influence of refolding kinetics on amyloid formation by transthyretin (TTR) variants was studied. TTR is a homotetramer and one of the many proteins known to be involved in human amyloid diseases. Numerous studies showed that dissociation of the native tetrameric structure into partially unfolded monomeric species precedes amyloid formation. Since the small structural differences observed in the crystal structures of TTR variants do not seem to justify their varying amyloidogenic potential, a significant effort has been devoted to search for thermodynamic and kinetic factors that may play a critical role on TTR stability, in order to fully understand the molecular mechanism of amyloid formation by TTR. Here, we have performed refolding kinetics assays, using intrinsic tryptophan fluorescence, with WT-TTR and its most common amyloidogenic variant V30M-TTR, to investigate the potential role of refolding kinetics on amyloid formation by TTR.