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Microsecond Dynamics of Macromolecules

It is generally believed that the biological macromolecules have motions ranging from ps to days and weeks and that these motions are essential for their function. The pico-nanosecond time window has been accessible using fluorescence, NMR and ESR while stop-flow techniques have opened ms and slower dynamics. However, till recently there were no experimental tools to study the microsecond time scale. The recent advances in stop-flow instrumentation, NMR, temperature jump and faster correlation spectroscopy measurements have made the microsecond window accessible experimentally.

We are using μs MLC and μs lanthanide probes as donors in FRET experiments to characterize conformational dynamics in microsecond time window. The method provides both the KINETICS and CONFORMATIONAL DISTRIBUTIONS as well as intra and inter molecular diffusion in ms to ms time. It is an EQUILIBRIUM method which can be applied to very diverse problems.

MAIN AIMS

  • Folding/Unfolding of protein secondary structure elements alpha-helix and beta-hairpin
  • Inter-domain motions in histidine and glutamine binding proteins
  • Lipid composition and phase state dependence of lateral diffusion in membranes
  • Lateral diffusion in biological membranes and domains such as RAFTs
  • Domain motions/ arm flexing in nucleic acid structures such as t-RNA, ribozyme, junctions and hairpins.
MDM

References

  1. Resolution of Multi-Exponential Spectral Relaxation of Yt-Base by Global Analysis of Collisionally Quenched Samples. Szmacinski, H., Gryczynski, I. and Lakowicz, J.R. (1996). J. Fluoresc., 6(3):177-185.
  2. Fluorescence Anisotropy Decays of 4-Cyano-N,N-dimethylaniline in Propylene Glycol Studied by Frequency-Domain Fluorometry. Kawski, A., Piszczek, G., Gryczynski, I., and Gryczynski, Z. (1998). Dedicated to J. R. Lakowicz on the occasion of his 50th birthday, Z. Naturforsch. 53a:711-716.
  3. Resonance Energy Transfer Study Using a Rhenium Metal-Ligand Lipid Conjugate as the Donor in a Model Membrane, Li, L., Gryczynski, I., and Lakowicz, J. R. (1999). Chem. Phys. Lipids 101:243-253.  
  4. β-Glycosidase from the Hyperthermophilic Archaeon Sulfolobus Solfataricus: Structure and Activity in the Presence of Alcohols, D'Auria, S., Nucci, R., Rossi, M., Bertoli, E., Tanfani, F., Gryczynski, I., Malak, H., and Lakowicz, J. R. (1999). J. Biochem. 126:545-552.
  5. The β-Glycosidase from the Hyperthermophilic Archaeon Sulfolobus Solfataricus: Enzyme Activity and Conformational Dynamics at Temperatures Above 100 C, D'Auria, D., Nucci, R., Rossi, M., Gryczynski, I., Gryczynski, Z., and Lakowicz, J. R. (1999). Biophys. Chem. 81:23-31.  
  6. End-to-end Diffusion on the Microsecond Timescale Measured with Resonance Energy Transfer from A Long-Lifetime Rhenium Metal-Ligand Complex, J. R. Lakowicz, R. Nair, G. Piszczek, and I. Gryczynski (2000). Photochem. & Photobiol. 71(2):157-161.  
  7. The Thermophilic Esterase from Archaeoglobus fulgidus: Structure and Conformational Dynamics at High Temperature, S. D'Auria, P. Herman, J. R. Lakowicz, E. Bertoli, F. Tanfani, M. Rossi, and G. Manco (2000). Proteins: Structure, Function and Genetics 38:351-360.  
  8. Perturbation of Conformational Dynamics of ASCUT-1 from Ascaris Lumbricodes by Temperature and Sodium Dodecyl Sulfate, D'Auria, S., Bazzicalupo, P., Rossi, M., Gryczynski, I., and Lakowicz, J. R. (2000). J. Fluoresc. 10(1):27-33.  
  9. Donor Fluorescence Decay Analysis for Energy Transfer in Double-Helical DNA with Various Acceptor Concentrations, Murata, S., Kusba, J., Piszczek, G., Gryczynski, I., and Lakowicz, J. R. (2000). Biospectroscopy 57:306-315.  
  10. Microsecond Dynamics of Biological Macromolecules, Lakowicz, J. R., Gryczynski, I., Piszczek, G., Tolosa, L., Nair, R., Johnson, M. L. and Nowaczyk, K. (2000). Methods in Enzymology, Academic Press, New York, 323:in press.
  11. The Esterase From the Thermophilic Eubacterium Bacillus Acidocaldarius: Structural-Functional Relationship and Comparison with the Esterase from the Hyperthermophilic Archaeon Archaeoglobus Fulgidus, D'Auria, S., Herman, P., Lakowicz, J. R., Tanfani, F., Bertoli, E., and Rossi, M. (2000). Proteins 40(3):473-481.  
  12. On Spectral Relaxation. Lakowicz, J. R. (2000). Photochem. Photobiol., 72(4):421-437.

Book Chapters

  1. Fluorescence Spectroscopy of Biomolecules. (1995). In: Molecular Biology and Biotechnology (R.A. Meyers, Ed.), VCH Publishers, Inc