Molecular structure of troponin C from chicken skeletal muscle at 3Å resolution

Troponin C is the Ca²⁺-binding subunit of the troponin complex and is involved in the calcium control of muscle contraction. The X-ray structure of chicken TnC has been determined at 3Å resolution using a single heavy atom derivative and application of a novel phase improvement and phase extension procedure. The protein has an unusual dumbbell-shape with a length of about 70A. The N- and C-domains are connected by a single long α-helix of about 9 turns. Two metal binding sites (the Ca²⁺-Mg²⁺ sites) in the C-domain are occupied by metal ions in the crystals and the helix-loop-helix Ca²⁺ -binding folds are very similar to those in other known Ca²⁺ -binding proteins. In contrast, the Ca²⁺ -specific sites in the N-domain appear unoccupied and the two putative Ca²⁺ -binding folds have a vastly different structural arrangement. The conformational rearrangements in the N-domain upon Ca²⁺ binding are believed to be the trigger for a cascade of protein-protein interaction alterations which lead to muscle contraction.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Muscle LIM protein plays both structural and functional roles in skeletal muscle

Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles. eccentric contractions; passive tension     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (>500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoresic dye front. Inclusion of 10 mm 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mm 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO4(2-) as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC2 resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca2+] by as much as 0.2 pCa units after LC2 extraction. At low concentrations of Ca2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC2 to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC2. LC2 extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.     

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.     

Studies on titin PEVK peptides and their interaction

Experiments were conducted on several synthetic and expressed peptides from the PEVK region of titin, the giant muscle protein. Different secondary structure prediction methods based on amino acid sequence gave estimates ranging from over 70% alpha helical to no helix (totally disordered) for the polyE peptide corresponding to human exon 115. Circular dichroism (CD) experiments demonstrated that both the positively charged PPAK modules and the negatively charged PolyE repeats had similar spectral properties with disordered secondary structure predominating. Gel permeation chromatography showed that both PPAK and polyE peptides had 2-4 times larger Stokes radii than expected from their molecular mass. Mixtures of the oppositely charged titin peptides caused no change in apparent secondary structure as observed by circular dichroism or migration properties using native gel electrophoresis. Similarly addition of calcium did not alter the CD spectra or peptide electrophoretic mobility of the individual peptides or their mixtures. The properties of both the PPAK and polyE type peptides suggest that both had most of the characteristic properties to be classified as intrinsically disordered proteins.