Photochemical interconversion between the red-absorbing (Pr) and the far-red-absorbing (P fr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The Pr-to-Pfr transition commences with a rapid Z-to-E photoisomerization at the C 15AC16 methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C15-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CAC stretch and NOH in-plane rocking modes within 500 fs, reveal that initial distortion of the C 15AC16 bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to Pr in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C 15-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C15AC16 Z-to-E isomerization occurs on the excited-state surface.photochemistry ͉ photoisomerization ͉ photosensory proteins ͉ plant signal transduction ͉ time-resolved vibrational spectroscopy L ight sensing and signaling responses mediated by photoreceptors are critical for the survival and growth of all life forms. Phytochromes are a class of biliprotein photoreceptors found in plants, bacteria, and fungi that are capable of sensing red/far-red light via interconversion between red-absorbing (P r ) and far-redabsorbing (P fr ) forms ( Fig. 1) (1). Light absorption by phytochrome triggers a rapid and reversible Z-to-E isomerization of the C 15 AC 16 methine bridge between the C and D rings of its bilin chromophore (2). This photochemistry subsequently drives changes in protein conformation that lead to changes in gene expression that influence growth and development (1, 3). Temporal resolution of the structure of the bilin chromophore during the photoisomerization process is important, not only to unravel common themes underlying ultrafast dynamics of biological reactions, but also for designing synthetic light-harvesting systems with rapid response times, efficient sensing, and energy storage.In the past, ultrafast pump-probe electronic spectroscopy has been used to probe the excited-state dynamics of plant and cyanobacterial (Cph1) phytochromes. Such studies reveal that formation of the isomerized primary photoproduct Lumi-R, characterized by a red-shifted electronic absorption maximum at 700 nm, occurs 25-40 ps after excitation (4-10). The P r excited state exhibits multiexponential fluorescence decay dynamics with at least two lifetimes (10 ps and 45 ps) (5), thereby implicating the presence of at least two excited states. The two-state model for P r * decay has also received support from ultrafast transient absorption measurements on the plant phytochrome phyA, the cyanobacterial phytochrome Cph1, and the bacteriophytochrome Agp1, all of which exhibit two ...

Long QT syndrome–associated mutations in KCNQ1 most often destabilize the protein, leading to mistrafficking and degradation.

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling and X-ray crystallography, we determined that FtsB self-associates and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an inter-helical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer, and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo.

A model of the core of the yeast spindle pole body (SPB) was created by a Bayesian modeling approach that integrated a diverse data set of biophysical, biochemical, and genetic information. The model led to a proposed pathway for the assembly of Spc110, a protein related to pericentrin, and a mechanism for how calmodulin strengthens the SPB during mitosis.

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state.

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.myosin | cardiac/skeletal myopathies | molecular dynamics | coiled-coils | protein structure M uscle contraction is primarily driven by the interactions between actin and myosin and the associated ATP hydrolysis, but the long-range transmission of force is based on the intrinsic ability of both proteins to self-assemble into organized filaments. The myosin thick filament is a well-characterized bipolar structure. The central area, or bare zone, is