No abstract

The effect of troponin I and caldesmon on phalloidin‐rhodamine‐ and 1,5‐IAEDANS‐labelled actin in skeletal muscle ghost fibers was investigated by polarized fluorescence. Both these proteins inhibited the structural alterations in the actin monomer and the increase of flexibility of actin filaments occurring on binding of myosin heads, and their effects were potentiated by tropomyosin. This immobilization of the actin filament through troponin I and caldesmon seems to originate from restriction of the relative motions of the two domains within the monomer.

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, ''S1,'' of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4°C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this ''path of influence'' might be.S ome years ago, we reported on the possibility of emplacing a new intramolecular crosslink in myosin S1 (1, 2) by using a then newly invented reagent, dibromobimane (3). Extending this work has now led to results of interest beyond the original objective. Our early work, based on identifying N-terminal end groups, indicated that CNBr cutting generated an H-shaped fragment whose ''crossbar'' is a dibromobimane joining Cys-522 and Cys-707. In the present work, this identification is strengthened by amino acid sequencing from Phe-496 to Glu-509 (interrupted by Trp-510) and from Glu-687 to Leu-711, further establishing the identity of the two cited cysteines. It was noted by Burke (4) that, although he could confirm the 522-707 crosslink at room temperature, the link appeared impossible at 0°C. His observation stimulated us to study the temperature dependence of the crosslinking. As we report here, the reaction with Cys-707 is rapid and depends little on temperature, but the subsequent crosslinking with Cys-522, achievable within 30 min at 25°C, requires a week at 0°C. Crystallography (5, 6) corresponding to near 0°C (as do all current images of S1) indicates an observable interthiol separation of about 23.49 A, compared with the 8-Å length of bimane (the next Cys in the sequence is even farther, at 41.85 Å). This suggests that, after anchoring at Cys-707, the yet unreacted function of dibromobimane and Cys-522 may engage in a slow temperature-dependent search for one another or else are brought together by a structural transition not evident in 4°C images. Although measurements of the ADP-S1 affinity with native S1 conform with the classic LoweyLuck (7) measurements on intact myosin, it is clear that the affinities are about one-third less with crosslinked S1, or with S1 reacted with monobromobimane (which reacts only Cys-707). Additionally, attaching either bimane alters the nuclear magnetic resonance arising in the P-atoms of the bound ␤-phosphate. These somewhat disparate o...

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site-the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product-in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl-for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.Various modifications of myosin subfragment 1 (S-1) (including complexation with actin) modulate its ATPase rate, but the underlying mechanisms of modulation are unknown. This study looks for attendant active-site changes that may suggest a general mechanism. An immediate stimulus for this study was the S-1 modification reported most recently, the generalized lysine methylation (1) used to prepare myosin crystals for structural studies (2). White and Rayment (3) have reported that such methylation markedly increases the Mg2+-ATPase activity of S-1, in a manner kinetically similar to that produced by attaching a bulky group to 5). Do the seemingly disparate modulations such as those induced by generalized lysine methylation, modification of Cys-707, transition from pH 7.5 to 9.2, and many other agencies (6, 7) share a common feature with one another and perhaps with F-actin binding? Here we have studied three intentionally disparate modulations-methylation, alkylation of Cys-707, and the pH transition. Our efforts centered on two characterizations of the ATPase site, both deriving from an earlier observation (8) that, in the presence of an inoffensive concentration of the collisional fluorescence quencher acrylamide, a fluorescent nucleotide increased its emission upon binding to (being engulfed by) the ATPase site. By using this effect one can obtain the binding constant of the nucleotide to the site.Further, as Rosenfeld and Taylor (9) later showed, it is also possible to extract "Stern-Volmer quenching constants" of the nucleotide interacting with the site, thus measuring the access of the quencher to the bound nucleotide. These two measures-binding constant and Stern-Volmer constantexpress the physical state of the ATPase site and can be measured before ("control") and after modulating t...

An effective vaccine against AIDS is unlikely to be available for many years. As we approach two decades since the first identification of human immunodeficiency virus, type 1 (HIV-1), currently, only one subunit vaccine candidate has reached phase 3 of clinical trials. The subunit approach has been criticized for its inability to elicit effectively cytotoxic T-lymphocyte (CTL) response, which is felt by many to be needed for protection against HIV-1 infection. In subhuman primates, a live attenuated simian immunodeficiency virus (SIV) vaccine candidate, capable of inducing CTL, has been found to confer prophylactic immunity sufficient to prevent simian AIDS. Because replication competent (live) attenuated viruses could over time revert to virulence, such a live attenuated approach has largely been dismissed for HIV-1. Here, we describe the creation of constitutively dead conditionally live (CDCL) HIV-1 genomes. These genomes are constitutively defective for the Tat/TAR axis and are conditionally dependent on tetracycline for attenuated replication with robust expression of viral antigens. Our results suggest that CDCL genomes merit consideration as safer "live" attenuated HIV-1 vaccine candidates.