Professor of The Academy
103D Biology East
Department of Biology
Johns Hopkins University
3400 N. Charles Street
Baltimore, MD 21218-2685
Office 410 516-7298
Lab 410 516-7300
Departmental fax 410 516-5213
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 simlations 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 at the following URLs:
We hope that you enjoy this latest paper from our laboratory.
Toptygin, D., Woolf, T.B., and Brand, L. (2010) ”Picosecond Protein Dynamics: The Origin of the Time-Dependent Spectral Shift in the Fluorescence of the Single Trp in the Protein GB1”, J. Physical Chem. B., (electronic version published August 2010.)
Xu, J., Chen, J., Toptigin, D., Tcherkasskaya, O., Callis, P., King, J., Brand, L., and Kntuson, J. (2009). Femtosecond Fluorescence Spectra of Tryptophan in Human gamma D-Crystallin Mutants: Site-Dependent Ultrafast Quenching". J.Am. Chem. Soc. 131:16751-16757.
Betagna, A., Toptygin, D., Brand, L.,and Barrick, D.(2008). "The Effects of Conformatiional Heterogeneity on the Binding of the Notch Intracellular Domain to Effector Proteins: A Case of Biologically Tuned Disorder". Biochemical Society Transactions, 36(2), 157-166.
Chen, Jiejin, Toptygin, Dmitri, Brand, Ludwig and King, Jonathan.,(2008) "Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human gamma D-Crystallin Studied by Time-Resolved Fluorescence"., Biochemistry 47, 10705-10721.
Xu J, Toptygin D, Graver KJ, Albertini RA, Savtchenko RS, Meadow ND, Roseman S, Callis PR, Brand L, Knutson JR. (2006). Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc. J Am Chem Soc. 128(4):1214-21.
Toptygin, D., Savichenko, R.S., Meadow, N.D., Roseman S. and Brand, L.(2002). Effect of the solvent refractive index on the excited-state lifetime of a single tryptophan residue in a protein. Biophysics. J. 82: 425A
Toptygin, D., Savichenko, R.S., Meadow, N.D. and Brand, L. (2001). Homogeneous Spectrally and time-resolved fluorescence Emission from Single-Tryptophan of IIAGlc Protein. Journal of Physical Chemistry B, 105, 2043-2055 .
Toptygin, D.and Brand, L., (2000) "Spectrally and Time-Resolved Fluorescence Emission of Indole During Solvent Relaxation: A Quantitative Model. Chem. Phys. Lett. 322, 496-502
Ya. K. Reshetnyak , O. A. Andreev , J. Borejdo, D. D. Toptygin, L. Brand and E. A. Burstein (2000) " The Identification of Tryptophan Residues Responsible for ATP-induced Increase in Intrinsic Fluorescence of Myosin Subfragment 1". Journal of Biomolecular Structure and Dynamics. 18, 113-125
Nanda, V., Liang, S-M. and Brand. L. (2000). Hydrophobic clustering in acid-denatured IL-2 and fluorescence of a Trp NH...p H bond. Biochemical and Biophysical Research Communications 279, 770-778.