THE MAIN-CHAIN DYNAMICS OF THE DYNAMIN PLECKSTRIN HOMOLOGY (PH) DOMAIN IN SOLUTION - ANALYSIS OF N-15 RELAXATION WITH MONOMER DIMER EQUILIBRATION/

The backbone dynamics of the pleckstrin homology (PH) domain from dyna min were studied by N-15 NMR relaxation (R(1) and R(2)) and steady sta te heteronuclear N-15 {H-1} nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 mu M, and by molecular dynamics (MD) simulations. The analysis was performed us ing the model-free approach. The method was extended in order to accou nt for observed partial (equilibrium) dimerization of the protein at N MR concentrations. A model is developed that takes into account both r apid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. I nternal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S-2 similar t o 0.9) and from the local correlation time <100 ps. Of the four extend ed loop regions that are disordered in the NMR-derived solution struct ure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S-2 down to 0.2 to 0.3) subnanosecond to nanose cond time-scale motion. Reorientation of the loops beta 3/beta 4 and b eta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S-2 similar to 0.9 more typical of the protein core . These loops, however, are involved in much slower processes of motio n resulting in a conformational exchange on a microsecond to submillis econd time scale. The motions of the terminal regions (residues 1 to 1 0, 122 to 125) are practically unrestricted (S-2 down to 0.05, charact eristic times in nanosecond time scale), suggesting that these parts o f the sequence do not participate in the protein fold. The analysis sh ows a larger sensitivity of the N-15 relaxation data to protein microd ynamic parameters (S-2, tau(loc)) when protein molecular mass (tau(c)) increases. The use of negative values of the steady state N-15{H-1} N OEs as an indicator of the residues not belonging to the folded struct ure is suggested. The amplitudes of local motion observed in the MD si mulation are in a good agreement with the NMR data for the amide NH gr oups located in the protein core. (C) 1997 Academic Press Limited.