The molecular basis for diminished muscle function in acidosis: a proposal.

A testable molecular proposal for the effects of acidosis on skeletal and cardiac muscle is presented. It is based on fluorescence studies published in 1974, which provided evidence for carboxylates in an EF-hand Ca binding site having an abnormal pKa. This results in an H-bound Blocked substate in the 3-state model of muscle regulation whose contribution inhibits myosin binding in the pH 7 to 6 range.

The mechanism of thin filament regulation: Models in conflict?

In a recent article, Heeley et al. (2019. https://doi.

The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin.

We investigated the functional impact of α-tropomyosin (Tm) substituted with one (D137L) or two (D137L/G126R) stabilizing amino acid substitutions on the mechanical behavior of rabbit psoas skeletal myofibrils by replacing endogenous Tm and troponin (Tn) with recombinant Tm mutants and purified skeletal Tn. Force recordings from myofibrils (15°C) at saturating [Ca] showed that Tm-stabilizing substitutions did not significantly affect the maximal isometric tension and the rates of force activation (k) and redevelopment (k). However, a clear effect was observed on force relaxation: myofibrils with D137L/G126R or D137L Tm showed prolonged durations of the slow phase of relaxation and decreased rates of the fast phase.

The myosin-activated thin filament regulatory state, M⁻-open: a link to hypertrophic cardiomyopathy (HCM).

This review proposes a link between the hypertrophic (HCM) and restrictive cardiomyopathies caused by mutations in several sarcomeric thin filament proteins, and the open state of the three-state muscle regulation theory. The three characteristics of various muscle systems reconstituted from HCM mutated proteins (increased Ca(2+)-sensitivity, increased basal activity in the absence of Ca(2+), and decreased cooperativity) can be explained by the contribution of a myosin-induced open state (M (-) ), which elevates the basal activity and competes with the normal Ca(2+)-activated pathway. A model based on the three-state theory of regulation, shows how a change in the closed/blocked equilibrium caused by a mutation that weakens the binding of troponin I to tropomyosin-actin can produce the characteristics of HCM.

John Gergely (1919-2013): a pillar in the muscle protein field.

Dr. John Gergely passed away on July 26, 2013 after a long and distinguished career. His publications spanned 67 years.

Polymorphism in tropomyosin structure and function.

Tropomyosins (Tm) in humans are expressed from four distinct genes and by alternate splicing >40 different Tm polypeptide chains can be made. The functional Tm unit is a dimer of two parallel polypeptide chains and these can be assembled from identical (homodimer) or different (heterodimer) polypeptide chains provided both chains are of the same length. Since most cells express multiple isoforms of Tm, the number of different homo and heterodimers that can be assembled becomes very large.

Long-range effects of familial hypertrophic cardiomyopathy mutations E180G and D175N on the properties of tropomyosin.

Cardiac α-tropomyosin (Tm) single-site mutations D175N and E180G cause familial hypertrophic cardiomyopathy (FHC). Previous studies have shown that these mutations increase both Ca(2+) sensitivity and residual contractile activity at low Ca(2+) concentrations, which causes incomplete relaxation during diastole resulting in hypertrophy and sarcomeric disarray. However, the molecular basis for the cause and the difference in the severity of the manifested phenotypes of disease are not known.

The 3-state model of muscle regulation revisited: is a fourth state involved?

The 3-state model of muscle regulation has been useful in explaining the roles of Ca2+ and myosin heads in activation and relaxation of striated muscle contraction. However, there are some phenomena, which cannot simply be explained by the 3-state model. These include increased Ca2+-binding caused by strong-binding myosin heads and residual active force at low Ca2+ in the case of familial hypertrophic cardiomyopathy.

Tropomyosin is in a reduced state in rabbit psoas muscle.

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm.

Ca2+-dependent photocrosslinking of tropomyosin residue 146 to residues 157-163 in the C-terminal domain of troponin I in reconstituted skeletal muscle thin filaments.

The Ca(2+)-dependent interaction of troponin I (TnI) with actin.tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca(2+), TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction.

Conserved Asp-137 imparts flexibility to tropomyosin and affects function.

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat.

Role of the head-to-tail overlap region in smooth and skeletal muscle beta-tropomyosin.

Tropomyosin (Tm) is an alpha-helical, parallel, two-chain coiled coil which binds along the length of actin filaments in both muscle and non-muscle cells. Smooth and skeletal muscle Tms differ extensively at the C-terminus encoded by exon 9. Replacement of the striated muscle specific exon 9a-encoded C-terminus with that encoded by exon 9d expressed in smooth muscle and non-muscle cells increases the affinity of unacetylated alpha-SkTm for actin [Cho, Y.

Functional homodimers and heterodimers of recombinant smooth muscle tropomyosin.

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers.

Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility.

To obtain information about the interaction of tropomyosin (Tm) with actin associated with the regulatory states of the muscle thin filament, we used luminescence resonance energy transfer (LRET) between Tb(3+) as a donor and rhodamine as an acceptor. A novel Tb(3+) chelator, S-(2-nitro-5-thiobenzoate)cysteaminyl-DTPA-Cs124, was synthesized, which specifically labels Cys groups in proteins. With the Tb chelate as the donor and tetramethylrhodamine-5-maleimide as the acceptor, both bound to specific Cys groups of Tm, we obtained 67 A as the distance between Tm's across the actin filament, a much shorter value than that obtained from structural studies (72-86 A).

Myosin-induced movement of alphaalpha, alphabeta, and betabeta smooth muscle tropomyosin on actin observed by multisite FRET.

The interaction of the alphaalpha, betabeta, and alphabeta smooth muscle tropomyosin (Tm) isoforms with F-actin was systematically studied in the absence and in the presence of myosin subfragment 1 (S1) using multifrequency phase/modulation Förster resonance energy transfer (FRET). A Gaussian double distance distribution model was adopted to fit FRET data between a 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid donor at either Cys-36 of the beta-chain or Cys-190 of the alpha-chain and a 4-dimethylaminophenylazophenyl 4'-maleimide acceptor at Cys-374 of F-actin. Experimental data were obtained for singly and doubly labeled alphabeta Tm (donor only at alpha, only at beta, or both) and for doubly labeled alphaalpha or betabeta Tm.

A modulatory role for the troponin T tail domain in thin filament regulation.

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament.

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET.

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C.

Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence.

The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined. These Tms span from 4 to 7 actin subunits. Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively).

Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units.

Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin.

Inhibition of actin-myosin subfragment 1 ATPase activity by troponin I and IC: relationship to the thin filament states of muscle.

Troponin I (TnI) is the component of the troponin complex that inhibits actomyosin ATPase activity, and Ca(2+) binding to the troponin C (TnC) component reverses the inhibition. Effects of the binding of TnI and the TnI-TnC (TnIC) complex to actin-tropomyosin (actinTm) on ATPase and on the binding kinetics of myosin subfragment 1 (S1) were studied to clarify the mechanism of the inhibition. TnI and TnIC in the absence of Ca(2+) bind to actinTm and inhibit ATPase to similar levels with a stoichiometry of one TnI or one TnIC per one Tm and seven actin subunits.