The widespread distribution of α‐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, α‐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 (δ= 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 ɛ‐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.

Regulatory myosin light-chain genes of Caenorhabditis elegans.

“…This agrees well with the measured size (18,000 daltons) for the larger of two MLC size classes of proteins identified in C. elegans (22). The molecular sizes predicted from the DNA sequences may be overestimates, since certain MLCs are modified posttranslationally by removal of N-terminal amino acids (24,65). The smaller of the two size classes of C. elegans MLC proteins (16,000 daltons; 22) is likely to be the alkali MLC(s).…”

Size and Charge Requirements for Kinetic Modulation and Actin Binding by Alkali 1-type Myosin Essential Light Chains

“…All striated muscle Al-type ELCs that have been analyzed contain N-terminal Ala (most) or Pro (some cardiac isoforms) that have been post-translationally modified by methylation (15). 2 The recombinant proteins used in this study are not modified in any way (and have been compared with synthesized peptides with a free N terminus).…”

“…Previous NMR studies on the intact rabbit skeletal A1-type ELC have suggested the involvement of one or more Lys residues and the N-terminal trimethylalanine residue in actin binding (15)(16)(17). In this report, we show by NMR that the key residues involved in binding to actin are Ala 1 , Lys 3 , and Lys 4 at the N terminus of the Al-type ELC.…”

1H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin

“…Myosin was extracted from rabbit fast twitch muscle (longissimus dorsi) from New Zealand white rabbits and subfragment 1 (Sl-A1 and Sl-A2) isoenzymes derived from this by chymotryptic digestion and purified as in [9]. Actin was also prepared from this source [9].…”

“…a stiffened polymer chain. The relevance of this structure to the role of this N-terminal segment in actin-myosin interaction [9] is described. The functional significance of the configuration adopted by segments of similar amino acid composition is then discussed in the context of their respective proteins of origin.…”