The objective of this proposed work is a quantitative understanding of protein dynamic structure. The approach to be used is the determination of fluorescence anisotropy decay of tryptophan residues and comparison of these experimental results with molecular dynamics calculations. The experimental quantities to be measured are the rate and amplitude of the tryptophan transition dipole reorientation as a function of temperature. The specific system chosen for study is a series of modified T4 phage lysozymes. This 18,700 dalton (164 residue) acetylmuramidase has a well-determined crystal structure with considerable similarity to HEW lysozyme. It is the subject of extensive study using a wide variety of physical methods. T4 lysozyme is available in a variety of """"""""mutant"""""""" forms, some containing only one of the three tryptophans at positions 126, 138 and 158. The other two sites may be replaced by tyrosine, serine or glutamine. Tyrosine substitution at positions 126 or 158 results in retention of full enzymatic activity. The Trp residues at positions 126 and 158 are at the protein surface; residue 138 is almost completely buried in the wall of the active site. A great variety of other amino acid substitutions are available in an existing mutant library. Many of these mutants are temperature sensitive and some have been proposed, on the basis of thermodynamic arguments, to result in large amplitude, low frequency protein motions. For this and other reasons, many of these substitutions are therefore expected to result in changes in the tryptophan indole group dynamics. The crystal structures of five of these mutant forms have been determined and found to be only slightly (and locally) different from that of the wild type. From the computational point of view, T4 lysozyme is sufficiently small that molecular dynamics simulations (using CHARMM) can be carried out into the time range of our fluorescence measurements. The test of these computational methods will be made by determining their ability to reproduce the rate and amplitude of the anisotropy decay as a function of temperature for several modified T4 lysozymes containing single trp residues. The effects of other perturbations (pH, guanidine and substrate binding) on trp dynamics will also be determined. The ability of the existing computational methods to correctly reproduce the equilibrium geometry of T4 lysozyme will also be investigated.
| Van Gilst, M; Hudson, B S (1996) Histidine-tryptophan interactions in T4 lysozyme: 'anomalous' pH dependence of fluorescence. Biophys Chem 63:17-25 |
| Harris, D L; Hudson, B S (1990) Photophysics of tryptophan in bacteriophage T4 lysozymes. Biochemistry 29:5276-85 |
