Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2ß2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Converter domain mutations in myosin alter structural kinetics and motor function.

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ß-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure.

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke.

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

Direct real-time detection of the structural and biochemical events in the myosin power stroke.

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)(2)FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

Condensed-phase effects on the structural properties of FCH2CN-BF3 and ClCH2CN-BF3: a matrix-isolation and computational study.

We have measured several IR bands of FCH2CN-BF3 and ClCH2CN-BF3 in solid nitrogen, argon, and neon. These bands include the B-F asymmetric stretch (νBF(a)), the B-F symmetric stretch (νBF(s)), the BF3 symmetric deformation or "umbrella" mode (δBF(s)), and the CN stretch (νCN). For both complexes, the frequencies of these modes shift across the various media, particularly the B-F asymmetric stretching band, and thus they indicate that the inert gas matrix environments significantly alter the structural properties of FCH2CN-BF3 and ClCH2CN-BF3. Furthermore, the frequencies shift in a manner that parallels the dielectric constant of these media, which suggests a progressive contraction of the B-N distances in these systems and also that it parallels the ability of the medium to stabilize the increase in polarity that accompanies the bond contraction. We have also mapped the B-N distance potentials for FCH2CN-BF3 and ClCH2CN-BF3 using several density functional and post-Hartree-Fock methods, all of which reveal a flat, shelflike region that extends from the gas-phase minimum (near 2.4 Å) toward the inner wall (to about 1.7 Å). Furthermore, we were able to rationalize the medium effects on the structure by constructing hybrid bond potentials composed of the electrostatic component of the solvation free energy and the gas-phase electronic energy. These curves indicate that the solvation energies are greatest at short B-N distances (at which the complex is more polar), and ultimately, the potential minima shift inward as the dielectric constant of the medium increases.

IR spectrum of CH3CN-BF3 in solid neon: matrix effects on the structure of a Lewis acid-base complex.

We have observed several IR bands of CH3CN-BF3 in neon and nitrogen matrices. For the 11B isotopomer in neon matrices, we observed the BF3 symmetric deformation band (nu7) as a doublet at 600 and 603 cm(-1), the BF3 symmetric stretching band (nu6) as a doublet at 833 and 838 cm(-1), the BF3 asymmetric stretching mode (nu13) at 1281 cm(-1) (partially obscured), and the C-N stretching mode (nu2) as a doublet at 2352 and 2356 cm(-1). The nitrogen matrix data are largely consistent with those reported recently, though we do propose a refinement of one band assignment. Comparisons of the frequencies of a few key, structurally sensitive vibrational modes either observed in various condensed-phase environments or calculated for two minimum-energy gas-phase structures indicate that inert matrix media significantly alter the structural properties of CH3CN-BF3. Specifically, the B-N dative bond compresses relative to the gas phase and other concomitant changes occur as well. Furthermore, the frequency shifts depict structural changes that occur across the various matrix hosts in a manner that largely parallels the degree of stabilization offered by these inert media.