Unfolding Domains in Smooth Muscle Myosin Rod

King, Lan; Seidel, John C.; Lehrer, Sherwin S.

doi:10.1021/bi00020a023

Cited by 9 publications

(12 citation statements)

References 23 publications

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“…The melting transition is reversible as observed after cooling and rescan. This result is comparable to that obtained with conventionally prepared LMM [15,16] and implies that LMMI and LMM2 have rather uniform structural stability along their length.…”

Section: Physicochemical Characterization Of the Recombinant Polypeptsupporting

confidence: 81%

Role of the C‐terminal extremities of the smooth muscle myosin heavychains" implication for assembly properties

et al. 1999

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The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their Cterminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.© 1999 Federation of European Biochemical Societies.helical sequences with different lengths. It has been suggested that these two tailpieces which are not present in cardiac or skeletal myosin heavy chains may differentially influence illament stability or packing (for review see [3]), or even the contraction velocity in vivo [4].To specify the role of the myosin heavy chain C-terminus extension in smooth muscle function we have prepared the two light meromyosin isoforms of rabbit (called LMM 1 and LMM2 in Fig. 1) by expression of their recombinant DNAs in Escherichia coli and compared various physicochemical properties of these two species.

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Section: Physicochemical Characterization Of the Recombinant Polypeptsupporting

confidence: 81%

Role of the C‐terminal extremities of the smooth muscle myosin heavychains" implication for assembly properties

et al. 1999

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“…A survey of previously characterized coiledcoils, however, reveals no correlation between helix length and thermal stability. Many structural proteins, including myosin, contain relatively long coiled-coil domains (in excess of 600 residues) but have relatively low melting temperatures (T m between 40 and 50°C) (67,68). Thus, although the coiled-coil domain of HSBP1 is similar in length to that of many proteins involved in transcriptional activation and signal transduction, unlike these domains, its thermal stability is comparatively lower and resembles that of the coiled-coil domains of structural proteins.…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Analysis of the Heat Shock Factor-binding Protein Reveals a Protein Composed Solely of a Highly Conserved and Dynamic Coiled-coil Trimerization Domain

Tai¹,

McFall²,

Huang³

et al. 2002

Journal of Biological Chemistry

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Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait. Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins. Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif. Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high ␣-helical content and moderate stability. NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases. Unlike previously characterized coiled-coils, backbone 15 N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region. Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range. NMR analyses suggest a rotationally symmetric trimer. Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.Protein oligomerization is a general mechanism for regulating and mediating diverse cellular processes including cytoskeletal interaction and cell motility, transcriptional activation, membrane trafficking, and intracellular signaling. A fundamental oligomerization motif is the "coiled-coil," which modulates protein structure and dynamics through its ability to mediate homotypic as well as heterotypic associations (for reviews of coiled-coils, see Refs. 1 and 2). Prominent examples of coiled-coil proteins include desmin and keratins forming the rigid framework of intermediate filaments (3), mannose-binding protein involved in antibody-independent immune response (4), Jun and Fos heterodimerizing to activate transcription (5), and the heat shock transcription factor 1 (HSF1) 1 oligomerizing temporally upon stress activation (6, 7). Because of its central role in biology and also because of its apparent simplicity, the coiled-coil motif has evoked considerable interest as a biological motif and as a paradigm for studies of protein folding (8 -10) and de novo protein design (11,12).The coiled-coil structural motif comprises two or more righthanded ␣-helices wrapped around each other with a small, left-handed superhelical twist (13). The hallmark of coiled-coils at the primary structure level is a seven-residue repeating sequence termed the heptad repeat in which the first and fourth residues (i.e. a and d in the sequence abcdefg) typically occupy the interhelical interface and are generally hydrophobic. Extensive structural and thermodynamic anal...

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“…Skeletal-muscle myosin rods exihibit multiple thermal-and denaturant-induced unfolding transitions involving regions of different stability [12,13]. Unfolding profiles of gizzard smoothmuscle myosin rod also show two thermaland denaturantinduced co-operative transitions, indicating the existence of two major structural domains [14]. In this study, we determined the number and size of unfolding domains of aortic rod and LMM and their relative stabilities against temperature by CD measurements.…”

Section: Introductionmentioning

confidence: 99%

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

King¹,

Jiang

Huang³

et al. 1995

Biochemical Journal

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We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.

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Unfolding Domains in Smooth Muscle Myosin Rod

Cited by 9 publications

References 23 publications

Role of the C‐terminal extremities of the smooth muscle myosin heavychains" implication for assembly properties

Role of the C‐terminal extremities of the smooth muscle myosin heavychains" implication for assembly properties

Structure-Function Analysis of the Heat Shock Factor-binding Protein Reveals a Protein Composed Solely of a Highly Conserved and Dynamic Coiled-coil Trimerization Domain

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

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