Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

The effect of nuclear radiation from the decay of uranium, thorium and their daughter elements on the specific gravity, unit-cell dimensions, and optical properties of zircon has been studied. During the course of the irradiation the specific gravity of zircon drops 16 %, the material becomes isotropic and so disordered as to fail to yield recognizable X-ray diffraction peaks. It is proposed that the observed effects are predominantly due to the displacement of atoms by recoil nuclei and by high temperatures generated in the path of nuclear particles. The breakdown of the structure is envisaged as a four-stage process in which the structure is first saturated with displacements; the saturated structure then breaks down into crystallites of ordered zircon which ultimately break down into a glass.

Upon photodissociation of its ligand, COHbA exhibits a wide range of nonequilibrium relaxation phenomena that start within a fraction of a picosecond and extend out to tens of microseconds. In addition, equilibrium fluctuations of the protein result in conformational averaging. All of these dynamics can have an impact on ligand rebinding. In an effort to better understand the relationship between conformational dynamics and ligand-binding reactivity, COHbA was embedded in a room temperature trehalose sugar glass (Hagen et al. Science 1995, 269, 959) in order to uncouple solvent motions from protein dynamics as well as reduce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kinetics show that the trehalose glass does not impede the initial fast relaxation of the iron-histidine linkage, but does dramatically impede conformational averaging and completely eliminates ligand escape at all temperatures from 140 K to room temperature. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetimes are nearly unchanged, but there is a complete absence of the nanosecond fluorescence decay (observed in aqueous solutions), which is replaced by a decay of ∼700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.