Folding and aggregation kinetics of a beta-hairpin.
We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.
Muñoz
Victor
V
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Ghirlando
Rodolfo
R
Blanco
Francisco J
FJ
Jas
Gouri S
GS
Hofrichter
James
J
Eaton
William A
WA
D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding
D010455:Peptides