Nuclear Magnetic Resonance Studies of Amino Acids and Proteins. Side-Chain Mobility of Methionine in the Crystalline Amino Acid and in Crystalline Sperm Whale (Physeter catodon) Myoglobin

We have obtained deuterium (²H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T₁) of l-[ϵ-²H₃] methionine, l-[ϵ-²H₃]methionine in a d, l lattice, and [S-methyl-²H₃] methionine in the crystalline solid state, as a function of temperature, in addition to obtaining ²H T₁ and line-width results as a function of temperature on [ϵ-²H₃]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering [Rothgeb, T. M., & Oldfield, E. (1981) J. Biol. Chem. 256, 1432–1446]. The results indicate that in the l-amino acid, methyl rotation having an activation energy (ΔE^⧧) of 8.3 ± 1 kJ dominates T₁ at low temperatures (≲−10 °C), while at higher temperatures an additional large-amplitude side-chain motion occurs which causes changes in the ²H NMR line shape and T₁. This motion is inhibited in the d, l lattice, indicating that lattice effects may have a strong effect on the mobility of anhydrous amino acids in the solid state. Further substitution at S^δ to form the sulfonium salt [S-methyl-²H₃]-methionine causes a large increase in ΔE^⧧, to 15.9 ± 2 kJ, a value comparable to the 14–16 kJ found in valine and leucine, which contain the structurally similar isopropyl moiety. These results suggest that the very low barriers to methyl rotation in the methionine side chain are due to long C-S bond lengths and the presence of only two substituents on sulfur, while the anomalous high-temperature behavior is due to a latticepacking effect. ²H T₁ results with methionine-labeled myoglobin are complex, reflecting the presence of fast large-amplitude side-chain motions, in addition to rapid methyl rotation. Our data indicate that Met-55 and Met-131 are motionally inequivalent in crystalline cyanoferrimyoglobin, in contrast to solution NMR results. We have also recorded ¹³C cross-polarization “magic-angle” sample-spinning NMR spectra of [ϵ-¹³C] methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. Cross-polarization transfer rates and proton rotating-frame relaxation time results again indicate that Met-55 and Met-131 are motionally inequivalent in the solid state, and the T_CH data indicate that Met-55 is more solidlike. However, we find that ¹³C chemical shifts in solution and those in the crystalline solid state are in very close agreement, suggesting that the average solution and crystal conformations are the same, in the area of Met-55 and Met-131.