Analysis of the backbone dynamics of interleukin-1.beta. using two-dimensional inverse detected heteronuclear nitrogen-15-proton NMR spectroscopy

Abstract

The backbone dynamics of uniformly 15N-labeled interleukin-1.beta. are investigated by using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. 15N T1, T2, and NOE data at a spectrometer frequency of 600 MHz are obtained for 90% of the backbone amide groups. The data provide evidence for motions on three time scales. All the residues exhibit very fast motions on a time scale of .ltorsim.20-50 ps that can be characterized by a single-order parameter with an average value of 0.82 .+-. 0.05. For a model comprising free diffusion within a cone, these residue-specific order parameters translate to an average cone semiangle of 20.7 .+-. 3.3.degree.. Thirty-two residues also display motions on a time scale of 0.5-4 ns, slightly less than the overall rotational correlation time of the protein (8.3 ns). These additional motions must be invoked to account for the discrepancy between experiment and the simplest theoretical formulation in which the internal motions are described by only two parameters, a generalized order parameter and an effective correlation time [Lipari, G., and Szabo, A. (1982a). J. Am. Chem. Soc. 104, 4546-4559]. In particular, while the simple formulation can account for the 15N T1 and T2 data, it fails to account for the 15N-1H NOE data and yields calculated values for the NOEs that are either too small or negative, whereas the observed NOEs are positive. With the introduction of two internal motions that are faster than the rotational correlation time and differ in time scales by at least 1-2 order of magnitude [Glore, G. M., Szabo, A., Bax., A., Kay, L. E., Driscoll, P. C., and Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991], all the relaxation data for these 32 residues can be fitted by two order parameters and an effective correlation time for the slower of the two internal motions. A simple model for these two motions is one in which the very fast motion involves axially symmetric diffusion within a core, while the slower motion comprises jumps between two different orientations of the NH vector. For such a model the jump angle (excluding the C-terminal residue) ranges from 15.degree. to 69.degree. with a mean value of 28.6 .+-. 14.0.degree.. Another 42 residues are characterized by some short of motion on the 30-ns-10-ms time scale, which results in 15N line broadening due to chemical exchange between different conformational substates with distinct 15N chemical shifts. In general, the motions on both the 0.5-4-ns and 30-ns-10-ms time scales are located in surface-accessible loops and turns connecting the .beta.-strands, as well as at the beginning and end of strands. In addition, some of the residues whose 15N line widths are exchange broadened are directly involved in backbone hydrogen bonding with bound internal water molecules or are in close proximity to residues that are. Finally, the kinetic and equilibrium properties of a slow conformational equilibrium between a major and a minor species, involving at least 19 residues and located on one contiguous face of the molecule, are characterized by using 1H-15N correlation spectroscopy, 1H-15N heteronuclear multiple quantum coherence-nuclear Overhauser enhancement spectroscopy, and 1H-1H nuclear Overhauser enhancement spectroscopy.