We have studied in detail the properties of the singular value decomposition of spectroscopic data sets which contain different types of measurement noise. The effects of totally random or """"""""shot"""""""" noise on the SVD are qualitatively different from those of """"""""wavelength- correlated"""""""" or baseline noise, and it is possible by post-processing the SVD to recover most of the signal content from a noisy data set only in the latter case. We have also characterized both experimentally and theoretically the effects of rotational diffusion on transient absorption measurements made with polarized photolysis and probe laser pulses. Procedures have been developed for obtaining properly isotropically averaged data sets from such measurements, which should allow determination of the conformational and ligand binding kinetics of hemoglobin with unprecedented accuracy. We have also derived oxygen binding curves for single crystals of human hemoglobin from polarized optical absorption spectra of crystals at various oxygen pressures. Hemoglobin in the crystal binds oxygen essentially non-cooperatively and with very low affinity, and these binding properties are essentially independent of pH (i.e., there is no Bohr effect). We have also performed large molecular dynamics simulations of the cooling of the photoexcited heme group in myoglobin in solution, and found that the dissipation of the excess heme energy in the protein in solution takes place only slightly faster than in the protein in a vacuum. We are also performing very long simulations of myoglobin in vacuo in order to study the orientational dynamics of the heme group in this protein to aid in the interpretation of anisotropy data from experiments using polarized excitation and probe pulses.
| Henry, Eric R; Bettati, Stefano; Hofrichter, James et al. (2002) A tertiary two-state allosteric model for hemoglobin. Biophys Chem 98:149-64 |
| Eaton, W A; Munoz, V; Hagen, S J et al. (2000) Fast kinetics and mechanisms in protein folding. Annu Rev Biophys Biomol Struct 29:327-59 |
| Eaton, W A; Henry, E R; Hofrichter, J et al. (1999) Is cooperative oxygen binding by hemoglobin really understood? Nat Struct Biol 6:351-8 |
