Detection of new temperature-dependent conformational transition in lysozyme by carbon-13 nuclear magnetic resonance spectroscopy.

Abstract

A specific temperature-dependent conformational transition of hen egg-white lysozyme, occurring between 20'C and 30'C in solution, has been detected by '3C-nuclear magnetic resonance spectroscopy. Selective changes in the chemical shifts of aromatic residues, together with differences in the chemical shifts, and nuclear Overhauser enhancement in the carbonyl, carboxyl, and alpha-carbon regions of the spectrum point to the vicinity of subsites D and E as the primary locus of the structural change.It has been recently shown that several proteins and enzymes can crystallize at temperatures higher than was usually assumed. Typical examples of temperature-dependent crystallization are yeast fatty acid synthetase (1), the Fab' Fragment from mouse IgA myeloma protein (2) and duck egg-white lysozyme 11 (3). The case of hen egg-white lysozyme has been extensively investigated over the past 2 years (4-6). It has been demonstrated that hen egg-white lysozyme crystals can be obtained between 250C and 60'C. These high temperature crystals (form B) are orthorhombic whereas, the classical crystals obtained in the temperature range from -36°C to 250 C and thoroughly studied by D. C. Phillips and coworkers (ref. 7 and references therein) are tetragonal (form A).Moreover, A crystals can convert into B crystals under the sole influence of temperature regardless of pH, ionic strength, protein concentration or nature of the precipitating salt (6). The only requirement for the occurrence of this transition is the presence of the liquid phase. In a typical experiment, A crystals obtained at low temperature are heated in presence of the mother liquor. At 220C-250C, A crystals dissolve and the formation of B crystals is observed. A and B crystals are present simultaneously at 250C but the B form is the only one that exists above 250C and up to 60'C. B crystals have been shown to be more stable than A crystals and, after dissolution, remain fully active against suspensions of Micrococcus luteus cells.In the light of these findings, it is reasonable to consider the possibility that the lysozyme molecule undergoes a conformational transition in the temperature range close to 250C which would account for the existence of the two crystalline forms. The observed dimorphism, could of course be accounted for by a crystal packing transition alone, but a significant conformational rearrangement of individual molecules would surely be reflected in a rearrangement of the lattice.Our aim, therefore, was to investigate limited specific changes in the three-dimensional arrangement of lysozyme in solution that occur in a rather narrow range of temperature corresponding to the crystalline phase transition.The structure of lysozyme in solution is known to undergo changes as a function of temperature. The reversible denaturation of lysozyme occurs between 60°C and 75°C, and has been studied extensively by McDonald and W. D. Phillips (8).The existence of two types of fluctuating unfolded forms D, and D2 has been proposed by Nakanishi et al. (9) on ...

Applications of natural-abundance nitrogen-15 nuclear magnetic resonance to large biochemically important molecules.

“…On the assumption that only dipolar relaxation to protons is important and that isotropic reorientation depends on the correlation time Tc, the dependence of NOE on -T, at 42 kG may be calculated (2) (3,4). These data are consistent with the assumption of dipolar relaxation for the amide nitrogen nuclei.…”

Comparisons of ring-current shifts calculated from the crystal structure of egg white lysozyme of hen with the proton nuclear magnetic resonance spectrum of lysozyme in solution

“…2 ppm from DSS, which decreased their conformation-dependent shifts by 0.25, 0.07, and 0.37 ppm between 10 and 66 OC. There was little evidence from a plot of chemical shift against temperature of a temperature transition in lysozyme at 25 OC, which has been proposed by Jardetsky and others [Cozzone et al (1975), but see Dobson (1975) and Shindo et al (1977)l to account for small shift changes seen in the I3C NMR spectrum of lysozyme. The effects of varying the concentration of lysozyme between 2.5 and 20 mM and the ionic strength between 0 and 0.83 by using sodium chloride in a solution of 5 mM lysozyme, both at pZH 5.0 and 54 OC, were negligible.…”

Determination of rotational correlation times of proteins in solution from carbon-13 spin-lattice relaxation measurements. Effect of magnetic field strength and anisotropic rotation

“…The a carbons of native proteins have yielded broad envelopes in reported 13C NMR spectra at low magnetic field strengths (14 and 23 kG). [1][2][3]5,6 Reported -carbon T\ values1,3,6 are not those of individual carbons but of the acarbon envelope. If the rotational motion is anisotropic, we must consider the possibility that the various contributing a carbons may not have identical T1 values (Figure 6).…”