We have investigated the site-specific folding kinetics of a photoswitchable cross-linked α-helical peptide by using single 13C=18O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6 °C), whereas at high temperatures (45 °C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained α-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an α-helical segment within a protein, rather than an isolated α-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.
BT - Proceedings of the National Academy of Sciences of the United States of America C1 - 0098 DA - 2008 Jul 15 DO - 10.1073/pnas.0712099105 IS - 28 J2 - Proc. Natl. Acad. Sci. U.S.A. LA - eng M3 - Research Article N2 -We have investigated the site-specific folding kinetics of a photoswitchable cross-linked α-helical peptide by using single 13C=18O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6 °C), whereas at high temperatures (45 °C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained α-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an α-helical segment within a protein, rather than an isolated α-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.
PY - 2008 SP - 9588 EP - 9593 T2 - Proceedings of the National Academy of Sciences of the United States of America TI - Alpha-Helix folding in the presence of structural constraints VL - 105 SN - 1091-6490 ER -