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

Abstract

Analysis by 'H-NMR spectroscopic techniques of the conformation of the N-terminal segment of the LC1 alkali light chain of rabbit skeletal muscle has shown that this portion of the molecule adopts a well-defined elongated configuration. T h s rod-like feature is a consequence of the Ala/Pro-rich composition and the functional aspects of such conformational preference in this and similar segments in other proteins are discussed.Studies of the dynamic aspects of protein structures have demonstrated the presence of internal motions over a broad time scale (1 ps to > 1 s [l]). Specific functional roles have been ascribed to both large-scale (domain) and more localized structural fluctuations [2] as distinct from the small-scale motions that occur universally in proteins (e. g. methyl group rotations). An obvious consequence of the occurrence of such structural flexibility is that the relevant segments of the protein do not possess a unique conformation in solution.Recent NMR investigations of a variety of protein systems, e. g. the dehydrogenase multienzyme complex have identified segments of these large organized structures with mobility substantially greater than that possessed by the rest of the polypeptide(s). In the case of the pyruvate dehydrogenase complex, the segment that possesses such mobility has been identified as deriving from the region of the primary structure that links the functional domains of the lipoate acetyltransferase (E2) core [7]. The observed mobility was ascribed to facilitating the movement of the lipoyl domains between the various active sites of the enzyme complex. A characteristic of the linkage between the E2 domains is the occurrence of a comparatively high content of alanine and proline. A comparable Ala/Pro-rich sequence is found at the N-terminal 'tail' of the A1 light chain (LCI) of the myosin head [8]. This segment is observed to contribute strongly to the mobile regions identified by the 'H-NMR studies of myosin and the myosin head [9].The physical properties ascribed to these segments, namely high conformational mobility and an apparent 'random-coil' configuration, are based on the NMR spectroscopic observaCorrespondence to B. A. Levine, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, England OX 1 3 QR Abbreviation. LCI, alkali light chain of skeletal myosin; NOE, nuclear Overhauser enhancement; COSY, two-dimensional homonuclear correlated spectroscopy; SDS, sodium dodecyl sulphate; S1, skeletal myosin subfragment-I. tion of segmental flexibility of sidechain groups independent of the rotational and translational motions of the entire molecule. Substantial mobility of the backbone of these segments has been inferred. The implication of extensive (9, w ) backbone torsion angle rotations and therefore an unstructured (random-coil) configuration has no experimental verification, however, since the NMR observations could arise from coordinated motion of a peptide of up to 50 amino acid residues in length. Insight into the conformation of the polype...

The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

“…Accordingly, the P1A1 sequence should exhibit a stronger PPII character since each Ala residue tends to assume the PPII conformation from the following Pro. Indeed, such a tendency was detected in NMR studies of the Pro‐rich N‐terminus of the light chain of skeletal myosin,53 which contains a (PA) 7 motif that exhibits an elongated conformation with the diffusion behavior of a rigid cylinder.…”

1H‐NMR spectroscopy of bovine lens β‐crystallin

“…In fact, one less alanine (from Ala199 of pB2) is present in the spectrum of the octomer compared to that of the trimer (Figs 7 and 5, respectively). NMR studies on myosin light chain [23], which also contains multiple Ala-Pro residues, concluded that these alternating amino acids result in a trans configuration for the proline, leading to a stiffened rod-like structure. Reduced flexibility of the N-terminal extension in pBl would decrease the likelihood of its resonances being resolved.…”

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

“…We have shown by deletion mutagenesis studies that the site lies within the first 11 residues and that actin binding by these residues results directly in modulation of the kinetic properties of S1 (11). The remaining residues of the N-terminal extension of A1-type ELCs are highly proline-rich and form an extended structure (12) responsible for correctly positioning the actin binding site on the surface of actin (13). In this study we address the role of individual amino acids of the human atrial A1-type ELC (HmAtELC; Ref.…”