High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase.

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

Rapid internal dynamics of BPTI is insensitive to pressure. (15)N spin relaxation at 2 kbar.

Pressure effects on the backbone dynamics of a native basic pancreatic trypsin inhibitor (BPTI) have been measured by (15)N spin relaxation and chemical shifts at 30 and 2000 bar. The experiments utilized the on-line variable pressure cell nuclear magnetic resonance system on (15)N-uniformly labeled BPTI at a proton frequency of 750.13 MHz at 36 degrees C. Longitudinal (R(1)) and transverse (R(2)) (15)N relaxation times and ((1)H)-(15)N nuclear Overhauser effects were measured for 41 protonated backbone nitrogens at both pressures. The model free analysis of the internal dynamics gave order parameters for individual H-N vectors at both pressures. The results indicate that rapid internal dynamics in the ps-ns range for the polypeptide backbone is not significantly affected by pressure in the range between 30 bar and 2 kbar. The result is consistent with the linear pressure dependence of (1)H and (15)N chemical shifts of BPTI, which suggests that local compressibilities and amplitudes of associated conformational fluctuation are nearly invariant in the same pressure range. Overall, we conclude that at 2 kbar BPTI remains within the same native ensemble as at 1 bar, with a small shift of population from that at 1 bar.

Ionophore properties of cationomycin in large unilamellar vesicles studied by 23Na- and 39K-NMR.

Cationomycin, isolated from Actinomadura azurea belongs to a large family of carboxylic polyether antibiotics, transporting monovalent cations through membranes by a mobile carrier mechanism, leading globally to an H+, M+ exchange. In this report the cation transporting properties of cationomycin were characterized in large unilamellar vesicles (LUVs) by 23Na- and 39K-NMR. Kinetic studies showed that cationomycin transported potassium more rapidly than sodium, and the more stable complex was formed with potassium at the water/membrane interface. The transport rate constants measured for cationomycin were compared with those obtained for monensin. Cationomycin transports Na+ more slowly than monensin and has a lower stability complex with Na+ because of the lower formation rate for the complex on the membrane surface. Our results show that transport selectivity of cationomycin is in favour of K+ versus Na+ while the reverse situation is observed for monensin. The relationships between the ionophore properties of cationomycin and monensin with their biological activities are discussed.