Ongoing improvements of genetically encoded fluorescent proteins have enhanced cellular localization studies and performance of biosensors, such as environmentally or mechanically sensitive fluorescence resonance energy transfer pairs, in cell biological and biophysical research. The brightest yellow fluorescent protein, widely used in these studies is YPet, derived from the jellyfish Aequorea victoria via the GFP derivative Venus. YPet dimerizes at concentrations used in cellular studies (KD1-2 = 3.4 μM) which impacts quantitative interpretation of emission intensity, rotational freedom, energy transfer, and lifetime. Although YPet is nearly 30% brighter than Venus, no atomic structures of YPet have been reported to ascertain the structural differences leading to the higher brightness, possibly due to the tendency to dimerize or oligomerize. Here, we report properties of a new YPet derivative, mCLIFY, a monomeric, bright, yellow, and long-lived fluorescent protein created by circular permutation of YPet and substitution of the amino acid residues thought to mediate dimerization. mCLIFY retains the advantageous photophysical properties of YPet but does not dimerize at least up to 40 μM concentration. We determined the atomic structure of mCLIFY at 1.57-Å resolution. Extensive characterization of the photophysical and structural properties of YPet and mCLIFY allowed us to elucidate the bases of their long lifetimes, enhanced brightness, and the difference in propensity to dimerize.

mCLIFY is a novel circularly permuted variant of the fluorescent protein (FP) YPet, a Venus-derivative. Using a battery of methods, we have shown that mCLIFY is exclusively monomeric retaining the favorable spectral and photophysical properties of the dimerizing YPet. We determined its atomic structure to 1.57 Å resolution and found that it is nearly identical to Venus, confirming that circular permutation did not alter its canonical structure. mCLIFY, YPet, and Venus are highly similar, only differing by six amino acids.

Fluorescent proteins (FP) have become widely used biophysical and cell biological tools that report on a variety of processes within cells. The popular yellow FP, YPet, serves as either a donor or acceptor for many FRET-based biosensors, yet it has been difficult to fully characterize. In studying our circularly permuted and equally bright variant, mCLIFY, features common to YPet were revealed, including β-barrel and chromophore sequence homology. We determined the atomic structure of mCLIFY to 1.6 Å resolution, which reveals a remarkably well-aligned π-interaction between the chromophore with Tyr37, as well as a water-mediated network of hydrogen bonds which supports the chromophore. Both features are likely promoting the robust photophysical properties of mCLIFY. To better understand the self-association properties of YPet, known to be a weak dimer, sedimentation velocity analytical ultracentrifugation (SV-AUC) determined a monomer-dimer dissociation constant KD of 3.4 μM, whereas mCLIFY did not dimerize. Size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering and multi-angle light scattering (SEC-SAXS-MALS) confirmed these findings. SAXS studies show that mCLIFY correlates well to the solution monomer, whereas YPET exists as an antiparallel dimer in solution. Their fluorescence intensities and anisotropies were compared via FLIM (Fluorescence Lifetime Imaging Microscopy) of FPs expressed in E. coli. Protein concentrations increased above 5 μM for both FPs within 45 minutes of IPTG induction. Unexpectedly, average anisotropy of YPet significantly decreased from 0.322 to 0.183 with increasing protein concentration presumably due to HOMO-FRET and/or trivial reabsorption of emitted photons. Anisotropy for mCLIFY (0.316) decreased at much higher concentrations. The data presented here suggest that potential quantitative errors from overexpression of non-monomeric FPs may be avoided with newly available, more fully studied monomeric variants like mCLIFY.

In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.

Although, currently available fluorescent proteins (FPs) provide an exceptional variety of high-performance options, new variations with improved properties are emerging. YPet is considered to be the brightest of the YFP variants, but it has the tendency to form dimers or oligomers under physiological conditions, compromising quantification of biophysical signals such as anisotropy and FRET. Here, we introduce mCLFY, a new monomeric bright yellow FP based on circular permutation of the amino acid sequence of YPet, separating the N- and C-termini of mCLFY from the protective barrel caps to potentially improve chromophore stability. The new N- and C-termini of mCLFY are located on the opposite end of the β barrel at YPet residues 175 in the loop arising from β-strand 8 and β-strand 9 residue 176. Fluorescence correlation spectroscopy (FCS) yielded a diffusion constant for mCLFY of D = 111 ± 1 μm2/s (s.e.m., n = 14), whereas for YPet, D = 57 ± 2 μm2/s (n = 14). Native gel electrophoresis supports these observations showing faster migration of mCLFY compared to YPet. Quantum yield (QY) of mCLFY is 0.76, using the reported 0.77 QY of YPet as a standard. Molar extinction coefficients were determined at λ = 517 nm, ε = 121 ± 7 x 103 and 124 x 103 M−1·cm−1 for mCLFY and YPet, respectively. mCLFY shows reduced pH sensitivity of brightness relative to YPet. Structural details that lead to the improved stability of mCLFY require further investigation. Using time correlated single photon counting, we determined fluorescence lifetimes of mCLFY and YPet to be 3.29 ± 0.04 ns and 3.27 ± 0.09 ns (n = 8 ea.), respectively. mCLFY is an improved FP similar to YPet, but with less tendency to dimerize and less pH sensitivity.

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5μm). Changes in the hs strain were measured by imposing length steps or small 4kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3–20mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8±1.1 kPa nm−1. Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7±3.7 kPa nm−1. An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (rF) is 23.5±1.0s−1 in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6±0.9s−1 at 20mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1ms) delay between generation of stiffness and generation of force by the cross-bridges.

The "conventional" isoform of myosin that polymerizes into filaments (myosin II) is the molecular motor powering contraction in all three types of muscle. Considerable attention has been paid to the developmental progression, isoform distribution, and mutations that affect myocardial development, function, and adaptation. Optical trap (laser tweezer) experiments and various types of high-resolution fluorescence microscopy, capable of interrogating individual protein motors, are revealing novel and detailed information about their functionally relevant nanometer motions and pico-Newton forces. Single-molecule laser tweezer studies of cardiac myosin isoforms and their mutants have helped to elucidate the pathogenesis of familial hypertrophic cardiomyopathies. Surprisingly, some disease mutations seem to enhance myosin function. More broadly, the myosin superfamily includes more than 20 nonfilamentous members with myriad cellular functions, including targeted organelle transport, endocytosis, chemotaxis, cytokinesis, modulation of sensory systems, and signal transduction. Widely varying genetic, developmental and functional disorders of the nervous, pigmentation, and immune systems have been described in accordance with these many roles. Compared to the collective nature of myosin II, some myosin family members operate with only a few partners or even alone. Individual myosin V and VI molecules can carry cellular vesicular cargoes much farther distances than their own size. Laser tweezer mechanics, single-molecule fluorescence polarization, and imaging with nanometer precision have elucidated the very different mechano-chemical properties of these isoforms. Critical contributions of nonsarcomeric myosins to myocardial development and adaptation are likely to be discovered in future studies, so these techniques and concepts may become important in cardiovascular research.