The contributions of protein motions to the dielectric response in solvent-inaccessible regions of calmodulin are examined. Two-pulse and three-pulse stimulated-emission experiments are used to examine the Stokes shift dynamics of coumarins in their ground and excited states. The results allow an evaluation of linear response theory. For coumarin 153, the ground-and excited-state solvation dynamics are similar to those for acetonitrile and methanol solvents. The solvation of coumarin 343 peptide in calmodulin is noticeably different in ground and excited states, indicating that the linear response picture is not appropriate in this case. The major difference is a more prominent fast component of solvation in the ground-state solvation dynamics. The ground-state energy gap correlation function is conjectured to be the best representation of protein relaxation, while that for the excited state seems to be influenced by the coupling of the protein modes to the large dipole created by excitation.

No abstract

The development of an optical trigger for protein folding is described. The optical trigger is an aryl disulfide embedded in a polypeptide such that the aryl disulfide constrains the peptide in a nonhelical conformation. Upon photocleavage of the SS bond, the peptide can then commence to fold into an R-helical conformation. Two thiotyrosine derivatives, (S)-4′-mercaptophenylalanine (Tty) and 3-N-(4′-mercaptophenyl)-(S)-2,3-diaminoproprionate (Aty), have been prepared and incorporated into polyalanine peptides. The ease of synthesis of protected forms of Tty and Aty amenable for solid phase synthesis, in four steps with 30% overall yield and six steps in 40% overall yield, respectively, make these attractive candidates as precursors of the optical trigger. CD and IR spectroscopy showed that the cyclic disulfide cross-linked peptides are much less helical than their linear counterparts. Following laser flash photolysis, peptide 7, which incorporates Tty, showed total recombination of the thiyl radicals within 1 ns. Peptide 16, which incorporates Aty, showed a significant amount of long-lived thiyl radicals from nanosecond to microsecond time scale. The process of recombination is hypothesized to be governed by the peptide conformation. Because of the significant amount of long-lived thiyl radicals generated from cyclic peptide 16, Aty should prove to be of general utility in the studies of protein folding on a time scale of sub-picoseconds and greater.

Pump-probe anisotropy measurements of HgI 2 photodissociation in ethanol were carried out. The reactive motion from electronically excited HgI 2 toward HgI and I is clearly evident in the signal. The subsequent randomization of the HgI orientational distribution occurs in the biphasic manner typical for some other diatomic molecules, with a 500-fs component that has an inertial motion contribution and a slow (8-10-ps) diffusive component. Interpretations of these regions were assisted by molecular dynamics simulations, which also were used to show that the rotational energy relaxation would be very fast in this system. The magnitudes of the anisotropies at different pump wavelengths show that the near-UV absorption of HgI 2 involves overlapping 1 Σ + f 1 Σand 1 Σ + f 1 Π transitions, consistent with magnetic circular dichroism (MCD) measurements. The anisotropy decay shows oscillations which are proposed to arise from oscillations in the volume and moment of inertia as the vibrational wavepacket moves in the anharmonic HgI potential.