Back to Table of Contents

43423-AC4
Thermodynamic Profiles of Fast Folding Peptides

Randy W. Larsen, University of South Florida

The mechanism through which heterogeneous biopolymers fold into specific catalytically active structures is one of the focal points of molecular biophysics. Knowledge of how such processes occur will not only help to understand the molecular basis for a wide range of diseases but will also lead to the design and synthesis of novel biocatalysts. The proposed project utilizes photothermal techniques to obtain ΔH and ΔV profiles for fast secondary structure folding associated with small water soluble peptides and proteins. The specific aims of this project are tto construct thermodynamic and kinetic profiles of (1) photo-triggered helix/coil transitions in small peptides using photothermal methods, (2) photochemicaly triggered folding in β-sheet and β-turn model peptides, and (3) secondary structure folding in the villin head-piece and leucine zipper (GCN-4) peptides.

PolyGlu20 pH Jump Folding: We have now completed Specific Aim I which involves using time-resolved photoacoustic calorimetry (PAC) to investigate ΔV and ΔH changes associated with the coil to helix transition in a synthetic (Glu)20 homopeptide in aqueous solution, both in the presence and absence of the denaturant guanidine hydrochloride (GdnHCl). At low pH's (< 4) PolyGlu20 undergoes protonation resulting in neutralization of the side chains leading to an increase in helical content due to reduced columbic repulsion. The coil to helix transition was found to occur with a rate of ~400 ns at 20^oC consistent with helix to coil transitions in non-charged peptides probed using laser T-jump methods. These studies also reveal significant effects due to denaturants which not only alter the kinetics (reducing the overall slow phase kinetics) but also significantly altering the thermodynamics of the folding reaction. A draft manuscript describing these results is now complete. We anticipate submission of the manuscript to J. Chem. Phys. B within the next month. GCN4 Folding: We have previously examined a synthetic system based upon a modified leucine zipper coiled coil system, GCN4. This 33 amino acid peptide adopts a stable dimmer configuration with each monomer having predominately α-helical secondary structure while the monomers are predominately random coil. We have now synthesized a 33 residue GCN4 peptide with Glu residues at position 15. Our plan is to poise this peptide at a pH slightly higher than the pKa of the Glu residues which should favor the unfolded monomers. oNBA will then be utilized to initiate the folding by protonating the Glu residues. Both PAC/PBD will be utilized to probe the folding thermodynamics similar to experiments described for the ‘caged' GCN4 system examined previously.

β-Amyloid Peptides: As an extension of the proposed work we are also looking into pH induced amyloid formation from the β-peptide. It has been suggested that ionizable groups on the β-peptide can enhance amyloid plaque formation and that the extrinsic pH may play an important role in this process. We have now completed the synthesis of a α-(1-28) sequence (N-AEFRHDSGYEVHHQKLVFFAEDVGSNK-C) which has been shown to undergo a pH dependent α-helix to β-sheet rearrangement with a pKa of >5.3. Thus, this system is ideal of the pH-jump/PAC-PBD protocols used to probe the polyGlu20 homopeptide coil to helix transition. In these experiments the time-resolved thermodynamics of the reverse, β-sheet to α-helix transition will be explored by poising the solution at pH ~ 6 and using a laser indeuced pH jump to reduced the solution pH to 4.5 thus initiating the rearrangement. Our time resolved photothermal methods will be used to probe the kinetics and thermodynamics associated with the aggregation process.

Folding of Cytochrome c: In addition to the small peptide folding systems we have also begun to examine the fast folding of larger protein system. The protein system is horse heart cytochrome c (hhCc) since the structure and slow folding dynamics of this protein have been well characterized. We are currently exploring two photo-initiation systems for hhCc folding. The first is a derivative of hhCc in which the native Met80 has been oxidized to a sulphoxide. We have now begun to examine the kinetics and themodynamics of CO photodissociation and subsequent rebinding to chloramine-T modified cytochrome c (CTCc) at high pH by transient absorption (TA) spectroscopy and PAC. The second photo-triggering system for hhCc involves the photolysis of CO hhCc at high pH. Previous studies have indicated that an unfolded state of hhCc can be prepared at pH ~ 12.9 in the presence of CO while the folded form of the protein dominates in the absence of CO. Thus, photolysis of CO hhCc at this pH results in the transient formation of a five coordinate ferro heme with the protein secondary/tertiary structure in an unfolded conformation. Subsequent binding of axial ligands gives rise to various folding trajectories that depend upon the nature of the bound ligand. We have now initiated a study to examine the thermodynamics associated with CO photolysis from hhCc/CTCc at ph 12.5 and pH 12.9.

Back to top