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A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

The SHAPES method for lead generation by NMR is useful for identifying potential lead classes of drugs early in a drug design program, and is easily integrated with other discovery tools such as virtual screening, high-throughput screening and combinatorial chemistry.

A method is proposed for direct mapping of spectral density functions of the rotational motions of H-X bond vectors, such as 'H-"N, by measuring a set of NMR relaxation parameters. The well known and frequently measured relaxation parameters T, and T, probe the spectral density function J(o) at five frequencies: 0, WN, wn, in -wN, and wu t wN. In this study, the longitudinal relaxation time T,( N,), the transverse relaxation times of in-phase coherence, T,( N,,Y), and of antiphase coherence, T2( 2H,N,,Y), the relaxation time of longitudinal two-spin order, T,(2H,N,), and the heteronuclear crossrelaxation rate bnN are measured for the heteronucleus N. These five relaxation parameters sample the spectral density function J(w) at the same five points where each measurement samples a subset of these frequencies with different weights. The five measurements permit an analytical calculation of J( w ) at these five frequencies. Since longitudinal proton relaxation plays a role in these relaxation parameters, a sixth measurement is necessary to determine this relaxation time. The theory and experimental techniques for measuring these relaxation parameters are discussed. Preliminary results of these techniques as applied to the 15N-enriched protein eglin care described. The proposed approach has the advantage that it does not rely on any a priori model assumptions about the shape of J(w); i.e., measurement of J(w) and interpretation can be separated. 0 1992 Academic PWS, 1~.Heteronuclear NMR relaxation times probe the motions of XH bonds (e.g., 13C-'H, "N-'H) in proteins through their dependence on the spectral density functions belonging to these bonds. The form of the spectral density functions, J(w), are determined by the fluctuations of the XH bonds with respect to the external magnetic field. Thus, the problem of characterizing the dynamics of the XH bond vectors reduces to the problem of characterizing the spectral densities. Current relaxation studies of proteins typically measure several parameters (e.g., T1, T2, and NOE) for the backbone "N and 13C nuclei (Z-5). While the measured relaxation rates are a function of J(w) at specific frequencies, they cannot determine what these values are (vide infra) . Thus, the spectral densities cannot be characterized experimentally using these measurements alone, and functional forms for J( w ) prescribed by theoretical models of motion must be used for further analysis. Most commonly used are the "wobbling-in-a-cone" model [see, for example, Woessner et al. (6), Kinoshita et al. ( 7), Richarz et al. (8) or the so-called "model-free approach" of Lipari and Szabo ( 9)]. Woessner's "wobbling-in-$ To whom correspondence should be addressed. 0022-2364192 $5.00 CoWright 0 1992 by Academic Press, Inc. AU rights of reproduaioa in any form reserved. 308 SPECTRAL DENSITIES FROM HOMONUCLEAR MEASUREMENTS 309 a-cone" model dominated the relaxation time research until 1982, while the Lipari and Szabo model has had this function for the last decade. Both approaches make an a priori assumpt...