Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Experimental and Theoretical Models to Probe Mechanisms of Biological Charge Flow

Nature is challenged to move charge efficiently over many length scales. From sub-nm to μm distances, electron-transfer proteins orchestrate energy conversion, storage, and release both inside and outside the cell. Uncovering the detailed mechanisms of biological electron-transfer reactions, which are often coupled to bond-breaking and bond-making events, is essential to designing durable, artificial energy conversion systems that mimic the specificity and efficiency of their natural counterparts. Here, we use theoretical modeling of long-distance charge hopping (Chapter 3), synthetic donor-bridge-acceptor molecules (Chapters 4, 5, and 6), and de novo protein design (Chapters 5 and 6) to investigate general principles that govern light-driven and electrochemically driven electron-transfer reactions in biology. We show that fast, μm-distance charge hopping along bacterial nanowires requires closely packed charge carriers with low reorganization energies (Chapter 3); singlet excited-state electronic polarization of supermolecular electron donors can attenuate intersystem crossing yields to lower-energy, oppositely polarized, donor triplet states (Chapter 4); the effective static dielectric constant of a small (~100 residue) de novo designed 4-helical protein bundle can change upon phototriggering an electron transfer event in the protein interior, providing a means to slow the charge-recombination reaction (Chapter 5); and a tightly-packed de novo designed 4-helix protein bundle can drastically alter charge-transfer driving forces of photo-induced amino acid radical formation in the bundle interior, effectively turning off a light-driven oxidation reaction that occurs in organic solvent (Chapter 6). This work leverages unique insights gleaned from proteins designed from scratch that bind synthetic donor-bridge-acceptor molecules that can also be studied in organic solvents, opening new avenues of exploration into the factors critical for protein control of charge flow in biology.