Alternate splicing of dysferlin C2A confers Ca²âº-dependent and Ca²âº-independent binding for membrane repair.

Dysferlin plays a critical role in the Ca²âº-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²âº and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1). Interestingly, these isoforms have markedly different responses to Ca²âº and phospholipid. Structural and thermodynamic analyses are consistent with the canonical C2A domain as a Ca²âº-dependent, phospholipid-binding domain, whereas C2Av1 would likely be Ca²âº-independent under physiological conditions. Additionally, both isoforms display remarkably low free energies of stability, indicative of a highly flexible structure. The inverted ligand preference and flexibility for both C2A isoforms suggest the capability for both constitutive and Ca²âº-regulated effector interactions, an activity that would be essential in its role as a mediator of membrane repair.

Negative coupling as a mechanism for signal propagation between C2 domains of synaptotagmin I.

Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission.

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803.

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.