Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.
We present an evaluation of the accuracy and precision of relaxation rates calculated using a variety of methods, applied to data sets obtained for several very different protein systems. We show that common methods of data evaluation, such as the determination of peak heights and peak volumes, may be subject to bias, giving incorrect values for quantities such as R1 and R2. For example, one common method of peak-height determination, using a search routine to obtain the peak-height maximum in successive spectra, may be a source of significant systematic error in the relaxation rate. The alternative use of peak volumes or of a fixed coordinate position for the peak height in successive spectra gives more accurate results, particularly in cases where the signal/noise is low, but these methods have inherent problems of their own. For example, volumes are difficult to quantitate for overlapped peaks. We show that with any method of sampling the peak intensity, the choice of a 2- or 3-parameter equation to fit the exponential relaxation decay curves can dramatically affect both the accuracy and precision of the calculated relaxation rates. In general, a 2-parameter fit of relaxation decay curves is preferable. However, for very low intensity peaks a 3 parameter fit may be more appropriate.
Abstraet. Single crystals of Dianin's inclusion eompound with methyl and ¡ deuterated p-xylene guest molecules were grown and studied by FT deuteron NMR. The spectra from the deuterated methyl groups reveal that these groups reo¡ rapidly down to 12 K; thereat~er they enter hato the tunneling regime. The rings of the p-xylene guests become motionless when T reaches 110 K. By measu¡ the orientation dependence of the quadrupole splittings, determining from these data the quadrupole eoupling tensors of the ring deuterons and relating these tensors to the C-D bond directions we infer the sites of the p-xylene guests in the cages of Dianin's inclusion compound. We final two sets of independent sites. Each contains three C 3 related individual sites. In each set the population of one of the sites is strongly depleted. The only large-angle molecular motions are 180 ~ rotational jumps about the long molecular axes. From measurements of T~ we conclude that these jumps are thermally activated, ro = 5.10 -~4 s, AE = 20 kJ/mol. Additional motions are rapid librations, also about the iong molecular axes. Their amplitude increases with increasing temperature, at 300 K it reaches 20 o. With 2D-exchange speetra we demonstrate that a p-xylene guest cannot change its site on a timescale of 100 ras anda tempe¡ expe¡ suggests that this is true on a timescale of several days even at T = 371 K.
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