Biomedical subjects
Publications and source records attributed to J A DiVerdi.
The motions of the aromatic amino acids of the fd bacteriophage coat protein are described by solid-state 2H, 13C, and 15N NMR. Tryptophan-26 is immobile on time scales as slow as 10(3) HZ. The phenylalanine and tyrosine rings undergo 180 degree flips about the C beta--C gamma bond axis more often than 10(6) HZ as well as small-amplitude rapid motions in other directions.
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31P NMR experiments on the filamentous bacteriophage fd are used to characterize the viral DNA. Because fd is a 16.4 X 10(6) dalton rod-shaped particle, methods of high-resolution solid-state NMR including cross polarization, proton decoupling, and magic angle sample spinning are utilized. The 31P chemical shielding tensor of solid fd is indistinguishable from that of single-stranded or double-stranded DNA in the absence of proteins; therefore the 31P chemical shift does not show evidence of structural changes in DNA upon incorporation into the virus. fd in solution has a very broad 31P resonance line width. The line width is due to static chemical shift anisotropy that is not motionally averaged, as shown by the generation of sidebands with magic angle sample spinning and a linear dependence of line width on magnetic field strength. These results indicate that DNA packaged inside fd is immobilized by the coat proteins.
31P NMR of native high molecular weight DNA shows that the phosphodiester backbone undergoes reorientation with a rotational correlation time of about 2 X 10(-6) s at 30 degrees C. This rate is consistent with the flexibility of the polymer and does not require the presence of internal phosphate motions. The line width of the phosphorus resonance of DNA is due to incompletely averaged 31P-1H dipolar couplings and 31P chemical shift anisotropy relaxation; high-power proton decoupling and magnetic field dependence experiments separate the two effects and allow the use of the line width for determination of the rotational correlation time. The line width is dependent on temperature, and an activation energy of 5-8 kcal/mol is calculated for the motion of DNA.
The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.