Conformational Dynamics of Biological Macromolecules

The interest of our laboratory is to better understand the dynamic structure of proteins, biological membranes and nucleic acids and to relate dynamics to function. Light provides a relatively non-invasive probe whose power lies in its ability to examine intact, functional macromolecular assemblies. Fluorescence spectroscopy is of value for studies of protein dynamics on the pico- to nanosecond time scales. We utilize single photocounting methods to measure the decay times of tryptophan in proteins as a function of emission wavelength. The data is used to generate time-resolved emission spectra. Similar data is obtained in the sub picosecond time scale using the upconversion method. The latter experiments are done at the NIH in collaboration with Dr. Jay Knutson. Suitable procedures are used to determine whether the ultrafast spectral shifts are due to microheterogeneity or to dielectric relaxation of the protein matrix and the solvent.

Although the depiction of proteins typically shown in publications, suggests a static configuration, this is a misleading representation. Proteins are moving on time scales from femtoseconds to seconds. Fluctuations on time-scales from femtoseconds to nanoseconds, although important to function are the least explored. We are using time-resolved fluorescence spectroscopy as described above in conjunction with molecular dynamics simulations to study ultra fast dynamic interactions in proteins.

Tryptophan and many other aromatics are solvatochromic. The dipole moment of Trp in the excited state is higher and has a different direction than in the ground state. Following excitation the Trp residue is out of equilibrium with its surroundings and will relax to the new equilibrium. Of particular interest is the relaxation of the protein matrix associated with motion of charged residues. This process is measured by obtaining fluorescence emission spectra as a function of time. The observed shift of the spectra to longer wavelengths reflects the relaxation process.

Although Trp red shifts in water are too fast to measure on the nanosecond time scale, as shown in Fig.3, time-dependent red shifts are observed for Trp in glycerol where the solvent relaxation is much slower.

Note that the x-axis shown above is in wavelength and is nonlinear and that the x axis shown below is linear and in wavenumbers.  Similar results (time-resolved emission spectra) are shown for the single Trp in the protein GB1 in Fig. 4

A logarithmic plot of the red-shift with time observed with the single Trp and also a single Fluoro Trp in GB1 Are shown in Fig. B below.

These relaxation curves are complex and more than one rate of relaxation is observed. Molecular dynamics simulations have been done on the relaxation processes about the Trp residue of GB1. Between 25 picoseconds and 2 nanoseconds all the relaxation is due to the motion of charged atom groups relative to the excited Trp residue.

Important questions that remain to be addressed are: What is the role of water relaxation at very early times? How important is heterogeneity? Is water associated with proteins (biological water) different in a fundamental way from normal water? How are these dynamic fluctuations related to substrate binding, protein folding and to enzyme catalysis. In collaboration with the laboratory of Professor Bertran Garcia-Moreno E., these studies are now being extended to mutant forms of staphylococcal nuclease.

Several of the questions raised above have now been answered for the protein GB1. The paper has been published in electronic format and may be accessed here:

Supporting information: Can be found here