We propose to continue our studies of the effects of increased hydrostatic pressure (0-10 kbar) on proteins, membranes and their interactions with other molecules. Pressures of this magnitude do not distort covalent bonds. Rather, such pressures perturb hydrophobic, electrostatic and hydrogen bonding interactions, which are the dominant forces determining the structure and function of macromolecules. These non-covalent interactions each display a different sensitivity to pressure, or stated equivalently, characteristic volume changes. Hence, the effects of pressure on the stability and/or association reactions of macromolecules may be used to infer the nature of the molecular interactions responsible for stability or binding. Fluorescence methods will be utilized, with a new emphasis on the variable-frequency phase-modulation fluorometer which is currently under construction. The following phenomena will be studied: 1) The effects of pressure on protein folding and dynamics, as revealed by the penetration of water-soluble quenchers and the time-dependent decays of tryptophan anisotropies. The latter will be determined by frequency-domain measurements. 2) The effect of pressure on the heme-binding site of myoglobin, as revealed by geometric reorientation of aryl anthracenes, and by the pressure-dependent binding of a fluorescence probe. 3) The effect of pressure on the partitioning of fluorophores and a fluorescent anesthetic into micelles and membranes. And 4) The effects of pressure on lateral phase separations in model membranes. Since the effects of pressure on proteins are smaller than on membranes we propose to construct a second pressure cell, capable of pressures to 10 kbar.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029318-06
Application #
3276864
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1981-02-01
Project End
1987-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
6
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Gryczynski, I; Eftink, M; Lakowicz, J R (1988) Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1. Biochim Biophys Acta 954:244-52
Lakowicz, J R (1988) Principles of frequency-domain fluorescence spectroscopy and applications to cell membranes. Subcell Biochem 13:89-126
Lakowicz, J R; Jayaweera, R; Joshi, N et al. (1987) Correction for contaminant fluorescence in frequency-domain fluorometry. Anal Biochem 160:471-9
Lakowicz, J R; Cherek, H; Gryczynski, I et al. (1987) Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics. Biophys J 51:755-68
Faucon, J F; Lakowicz, J R (1987) Anisotropy decay of diphenylhexatriene in melittin-phospholipid complexes by multifrequency phase-modulation fluorometry. Arch Biochem Biophys 252:245-58
Keating-Nakamoto, S M; Cherek, H; Lakowicz, J R (1987) Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies. Anal Chem 59:271-8
Lakowicz, J R; Laczko, G; Gryczynski, I (1987) Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry. Biochemistry 26:82-90
Lakowicz, J R; Laczko, G; Gryczynski, I et al. (1986) Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry. J Biol Chem 261:2240-5
Hermetter, A; Lakowicz, J R (1986) The aggregation state of mellitin in lipid bilayers. An energy transfer study. J Biol Chem 261:8243-8
Lakowicz, J R; Laczko, G; Gryczynski, I (1986) Picosecond resolution of oxytocin tyrosyl fluorescence by 2 GHz frequency-domain fluorometry. Biophys Chem 24:97-100

Showing the most recent 10 out of 11 publications