John Gergely

Zero-length crosslinking procedure with the use of active esters.

A two-step zero-length crosslinking procedure for studying protein-protein complexes has been developed. One component of a complex is briefly incubated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide resulting in the conversion of some of the protein carboxyls into succinimidyl esters. The reaction is stopped by addition of beta-mercaptoethanol and other interacting proteins are then added. Crosslinking arises from substitution of lysine epsilon-amino groups of these proteins for the succinimidyl moieties during a 1- to 2-h incubation period. The advantage of this method versus one-step zero-length crosslinking is that only one component of the complex is exposed to the crosslinker, which eliminates complications arising from the formation of crosslinks among several proteins of a multicomponent complex. Furthermore, crosslinks can be formed even in the presence of reagents, such as dithiothreitol and EDTA, that would interfere with direct crosslinking with EDC.

Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction.

Recent developments in the field of myofibrillar proteins will be reviewed. Consideration will be given to the proteins that participate in the contractile process itself as well as to those involved in Ca-dependent regulation of striated (skeletal and cardiac) and smooth muscle. The relation of protein structure to function will be emphasized and the relation of various physiologically and histochemically defined fiber types to the proteins found in them will be discussed.

Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin.

Abstract The two essential thiol groups of myosin, SH-1 and SH-2, have been localized in an ~ 20K segment of the heavy chain by analysis of the distribution of radioactivity after tryptic digestion of tryptic heavy meromyosin (HMM) or papain-HMM subfragment-1, both labeled at SH-1 and SH-2 with [14C]iodoacetamide and [14C]N-ethyl maleimide, respectively. The results are discussed in the framework of earlier work (Balint, M., Sreter, F. A., Wolf, I., Nagy, B., and Gergely, J. (1975) J. Biol. Chem. 250, 6168–6177) on the tryptic fragmentation of myosin heavy chain and in the light of more recent work on the location of a fragment that reacts with a photoaffinity analog of ATP (Szilagyi, L., Balint, M., Sreter, F. A., and Gergely, J. (1978) Fed. Proc. 37, 1695) and of suggestions concerning the binding of ATP in the region containing the SH-1 and SH-2 (Elzinga, M., and Collins, J. H. (1977) Proc. Nat. Acad. Sci. USA74, 4281–4284).

Structural and functional changes of myosin during development: Comparison with adult fast, slow and cardiac myosin

Abstract ATPase (Ca2+ and K+ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO4 polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC2 except that the relative amount of LC3 is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.

Molecular mechanism of troponin-C function

ConclusionsThere is now a large body of evidence in support of the view that Ca2+ binding to the low affinity sites of TnC induces a movement of helices B and C away from helices A and D, thus opening a hydrophobic cavity, the site of interaction with TnI. Another site of similar structure is formed by the helical segments in the C-terminal domain. Both sites appear to interact with the inhibitory segment of TnI. Whereas the interactions at both sites are necessary for the full regulatory activity of TnC, the interaction at the C-terminal domain stabilizes the complex and that involving the N-terminal domain is directly linked to the release of inhibition. In the absence of Ca2+ the inhibitory region of TnI would preferentially bind to actin and on Ca2+ binding to sites I and II it would switch to the site in the N-terminal domain of TnC. Detachment of TnI from actin gives rise to further events in thin filament regulation.

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-ai HMM and S-l are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S- 1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S- 1 prepared with papain in the presence of Mg2 + : the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the aetin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-a&in is controlled by a mode of rotation within the F-actin filaments.

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

Tryptic digestion and localization of calcium uptake and ATPase activity in fragments of sarcoplasmic reticulum

Fractions sedimented at 25,000 g for 50 minutes from homogenates of mouse hind leg muscles were prepared according to the procedure of Von Korff originally designed to obtain intact cardiac mitochondria (41). Negatively stained preparations of this fraction (fragmented sarcoplasmic reticulum) appear to be essentially free of contamination with mitochondrial fragments and myofilaments. Vesicles consisting of a globular head with a diameter of 0.1–0.2 μ and one or more tail-like portions with a roughly constant width of 350 and a varying length of 0.05–1 μ (most frequently 0.2 μ) predominate. The surface of the vesicle, both of the head and tail portion, is covered with particles approximately 40 in diameter and spaced 50–80 from each other, the center being 40–60 from the surface of the vesicular membrane. Ca deposits, in the presence of oxalate, were localized in the globular portion. ATPase activity, as judged electron microscopically from the deposition of lead phosphate, was also localized in the globular portion, very frequently at the junction with the tail. Digestion of vesicles with trypsin caused changes in both the Ca uptake and ATPase activity. With a trypsin-vesicular protein ratio of 1:25 to 1:100 a rapid drop in Ca uptake was accompanied by an increase in ATPase activity. With even lower (1:300) ratios, a rapid initial drop in Ca uptake was eventually followed by a slower phase of decrease; the ATPase in this case increased monotonically with an inflection point subsequent to the rapid drop in Ca2+ uptale. The digestion caused structural disruption of the vesicle, first in the tail, and eventually led to swelling of the globular portion and the appearance of areas devoid of regularly arranged particles on the membrane.

Fractionation of solubilized sarcoplasmic reticulum.

Summary A method has been developed which permits the separation of three protein components of the fragmented sarcoplasmic reticular membrane following solubilization with Triton X-100. One fraction contains a 100,000 dalton component and constitutes about 20–30% of the total membrane protein; in this fraction the level of ATPase activity and phosphorylated intermediate formation is about four times higher than the level after activation with Triton. Another fraction (62,000 dalton, 20–25% of the total protein) is precipitated upon addition of 4 mM Ca ++ . The third fraction (125,000–160,000 dalton) constitutes about 50% of the total protein.

The conformation of myosin during the steady state of ATP hydrolysis: Studies with myosin spin labeled at the S1 thiol groups☆

Abstract MgATP produces a large change in the ESR spectrum of myosin that has been spin labeled at the S1 thiol groups. This change, indicative of increased mobility of the label, persists during the steady state of the hydrolysis of ATP, and when ATP has been depleted the spectrum changes to one identical with that observed on adding MgADP. It appears that the binding or hydrolysis of ATP by myosin in the presence of Mg induces a conformational change in myosin that persists during the steady state. If, as proposed by Taylor and coworkers (1,2) the predominant species present during the steady state is the myosin-ADP complex, then the conformation of myosin in this complex depends on whether the complex was formed with added ADP or in the course of the hydrolysis of ATP.