The concerted movement of the switch region of Troponin I in cardiac muscle thin filaments as tracked by conventional and pulsed (DEER) EPR.

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C.

Full Atom Simulations of Spin Label Conformations.

Interpretation of EPR measurables from spin labels in terms of structure and dynamics requires knowledge of label behavior. General strategies were developed for simulation of labels used in EPR of proteins. The criteria for those simulations are (a) exhaustive sampling of rotamer space, (b) consensus of results independent of starting points, and (c) inclusion of entropy.

Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC).

Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores.