The widespread distribution of alpha-N-trimethylalanine as the N-terminal amino acid of light chains from vertebrate striated muscle myosins.

Abstract

Identical tripeptides of the sequence X-Pro-Lys, where X is an unknown blocking group, were isolated from trypsin digests of bovine cardiac alkali light chain and the LC2 light chain of rabbit fast muscle. Chemical, electrophoretic and 1H-NMR evidence characterized X as an unusual amino acid, alpha-N-trimethylalanine (Me3Ala), which was earlier reported as the N-terminal amino acid of the A1 alkali light chain of rabbit fast muscle [Henry et al. (1982) FEBS Lett. 144, 11-15]. The narrow line width and chemical shift position (delta = 3.23 ppm) of the--N+-(CH3) protons of Me3Ala made 1H-NMR spectroscopy a convenient method to search for this residue in other light chains. A survey of many different light chains showed that this signal was present in all vertebrate striated muscle light chains of the A1-type (LC1, 'essential' light chains) and LC2-type ('DTNB'-light chains, 'phosphorylatable' light chains) but was absent from all invertebrate muscle and vertebrate smooth muscle light chains tested. It was also absent from the vertebrate fast-muscle-specific A2-type (LC3) light chains. The spectral characteristics of these signals were consistent with their having arisen from the protons of an--N+-(CH3)3 grouping. Since no epsilon-trimethyllysine could be detected in acid hydrolysates of these proteins, it appears that Me3Ala is a general feature as the N-terminal amino acid in these light chains. 1H-NMR studies on bovine cardiac myosin subfragment 1 (S1) showed that the Me3Ala methyl proton signal was clearly visible and that the spectrum more closely resembled that of a rabbit S1 isoenzyme, S1(A1), than S1(A2), suggesting that the 40-residue N-terminal segment of the alkali light chain in cardiac S1 also possesses a high segmental mobility. Addition of actin caused the same gross changes to the cardiac S1 spectrum as noted earlier for rabbit S1(A1) [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. In particular, a marked reduction in the segmental mobility of the N-terminal region of the alkali light chain was noted, consistent with a direct interaction of this area with actin.