4188 BIOCHEMISTRY Protein Mobility and Self-Association by Deuterium Nuclear Magnetic Resonance? Jan B. Wooten and Jack S. Cohen* WOOTEN zyxw AND COHEN ABSTRACT: Hen egg white lysozyme has been prepared in which the C' position of the single histidine residue is sub- stituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (*H NMR) spectrum in H20 shows a broad resonance (500-1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [Shindo, H., Cohen, J. S., zyxwvut & Rupley, J. A. (1977) zyxwvutsrqpon Biochemistry 26, 38791. Correlation times calculated from spin-spin relaxation times (T,) derived from Spectroscopic studies of magnetic nuclei in biological systems have generally concentrated on those nuclei which provide the highest possible spectral resolution. Deuterium, because of its nuclear quadrupole moment, exhibits broad nuclear res- onances in slowly reorienting molecules and hence does not lend itself to high-resolution N M R studies. However, the particular properties of the deuteron can be taken advantage of in other respects; the intrinsic nuclear relaxation due to the quadrupole moment renders the deuteron an effective monitor of local mobility independent of other nuclei. This property has been extensively used to study deuterated membrane systems (Seelig, 1977), the binding to proteins of small molecules (Szilagyi et al., 1977; Andrasko & Forsen, 1974; Gerig & Rimerman, 1972; Zens et al., 1976), and selectively deuterated peptides (Glasel et a]., 1973), but there have been no direct applications to proteins. One reason for this fact is that the broadness of deuterium N M R signals precludes the resolution of multiple signals. Therefore, to obtain meaningful results from the application of this method to proteins it is necessary to deal with a singly deuterium-enriched molecule. We now report the first such study of mobility in a protein, hen egg white lysozyme, containing a single deuterium atom. Previously, only heme methyl groups of myoglobin have been enriched with deuterium (Oster et al., 1975). Lysozyme was chosen principally for two reasons: first, it contains a single histidine residue at position 15, and this could be selectively deuterated (Meadows et a]., 1968); and second, it self-asso- ciates as a function of pH in a manner in which the histidine residue is known to be involved (Shindo et al., 1977). Thus, this system provides a convenient model for the utility of deuterium NMR as a probe of macromolecular mobility. Experimental Procedures Materials. Hen egg white lysozyme (Worthington Bio- chemical Corp.) was exchanged at pH 9 in 99.7% D20 at 37 "C in a sealed tube under nitrogen. The substitution of the C' proton of His-15 with 2H was monitored by the disap- From the Developmental Pharmacology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205. zyxwvutsrqpon Received May 2, 1979. This work was presented at the 20th Experimental NMR Spectroscopy Conference, Asilomar, California, Feb 1979. the *H line widths indicate that His-1 5 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [t-2H]imidazole showed a small p H dependence of the spin-lattice relaxation time ( Tl), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance ('H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T, values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring. pearance of the IH resonance in the 220-MHz proton NMR spectrum. After 1 week, the solution was neutralized with 1 N DC1, lyophilized, and then lyophilized 3 times from 2H- depleted water (Aldrich) to remove residual D20. The zy [t- 2H] imidazole was prepared under similar conditions. 'H NMR Measurements. The 41.4-MHz 2H spectra of 1 1 and 7 mM solutions of lysozyme in 0.2 N NaCl were obtained at 25 "C on a home-built spectrometer equipped with a Bruker superconducting magnet. Typically, 20 000 scans were ob- tained by using a spectra window of 20 kHz. A 10-Hz ex- ponential line broadening was applied before Fourier trans- formation. A 180°-r-900 pulse sequence was used to partially suppress the sharp interfering resonance due to residual HOD, with zyxwvu T chosen such that the much broader lysozyme resonance was not disturbed. The line widths at half-height (W,,,) were obtained by fitting the observed spectra with Lorentzian lines using the Curve Analysis Program on the Nicolet 1180 data system. The 2H TI values of 0.5 M solutions of [€-*HI- imidazole were measured by using the standard inversion- recovery pulse sequence. The 2H chemical shifts were measured relative to the IH resonance of CD3CN (1%) in the aqueous solutions. Results The 2H N M R chemical shift of [t- 2HH]imidazoleis essentially the same as the IH shift of the normal imidazole, and the pK, value obtained, 6.960 zy f 0.007 (Figure I), agrees with that obtained from lH N M R spectra, 7.15 (Sachs et al., 1971). Addition of fairly high levels of paramagnetic Cu2+ion ( M) results in an additional pH inflection, which presumably reflects the formation of Cu-Iml complex (Sundberg & Martin, 1974). The 2H relaxation time, TI, also reflects the ionization of the imidazole ring with an apparent pK, = 6.69 f 0.09 (Figure 2). In the presence of CuZ+, the T, titration curve is somewhat altered, but the 'H resonance is readily observable even at this high Cu2+ ion concentration. [t-2H]His-15 Lysozyme. A typical 2H spectrum is shown in Figure 3 and consists of two resonances, that of the singly 2H-labeled protein and the residual HOD. Since there is such a difference in the line width between these signals, no dif- [r-2H]Zmidazole. ' Abbreviations used: Im, imidazole: tri-WAG, zyxw tri-N-acetylglucosamine. This article not subject to US. Copyright. Published 1979 by the American Chemical Society