Electrostatic coupling leading to conformational changes in proteins is challenging to demonstrate directly, it requires that both the local, discrete electronic details and dynamic information relevant to the functional descriptions are probed. Here, as a novel study to address this challenge, the roles of an aromatic residue in influencing the functional conformational changes of a membrane receptor in its natural membrane environment are reported. Previously intractable discrete electronic details have been obtained using 2D solid-state NMR of specifically labelled receptor, reinforced with molecular dynamic simulations, mutational analysis and functional assays, supported by and compared with rigid-atom crystal structural models. Hydrogen bonding and hydrophobic interactions are identified as the mechanistic origin for direct electromechanical coupling to the dynamics of conformational changes within the receptor.

Tyrosine 185 (Y185), one of the aromatic residues within the retinal (Ret) chromophore binding pocket in helix F of bacteriorhodopsin (bR), is highly conserved among the microbial rhodopsin family proteins. Many studies have investigated the functions of Y185, but its underlying mechanism during the bR photocycle remains unclear. To address this research gap, in situ two-dimensional (2D) magic-angle spinning (MAS) solid-state NMR (ssNMR) of specifically labelled bR, combined with light-induced transient absorption change measurements, dynamic light scattering (DLS) measurements, titration analysis and site-directed mutagenesis, was used to elucidate the functional roles of Y185 during the bR photocycle in the native membrane environment. Different interaction modes were identified between Y185 and the Ret chromophore in the dark-adapted (inactive) state and M (active) state, indicating that Y185 may serve as a rotamer switch maintaining the protein dynamics, and plays an important role in the efficient proton-pumping mechanism in the bR purple membrane.

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin-4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid-state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark-adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.

The carotenoid lycopene in photosynthetic organisms forms the critical precursor to the biosynthesis of reaction center carotenoids beta-carotene and lutein. In plants and cyanobacteria, a redox-controlled flavoenzyme carotenoid isomerase (CRTISO) catalyzes the four-step geometric isomerization of 7,9,9 0 ,7 0 -tetra-cis-lycopene (prolycopene) to all-trans-lycopene. In chloroplasts, the functional loss of CRTISO has been shown to be rescued by a light-mediated isomerization pathway. In order to address the chloroplastspecificity and compare the efficiency of the photoisomerization reaction against redox-controlled enzyme catalysis, we need to track the excited state dynamics of prolycopene, and evaluate the nature of electronic states that lead to the photoisomerization. Using broadband femtosecond transient absorption spectroscopy, we observe~610 fs rise of the triplet state from the photoexcited S 2 with a quantum yield of~0.19. The triplet state mediates the first C=C bond isomerization at symmetric 9 or 9 0 position on the tetracis backbone to yield the tri-cis-lycopene with 15% quantum yield. However, direct sensitization of the photoreactive triplet state via meso-tetraphenyl porphyrin sensitizer under steady state illumination leads to all-trans-lycopene with 58% quantum efficiency. Our work implies that long-lived chlorophyll triplets activate the efficient isomerization of prolycopene in chloroplasts in absence of CRTISO activity. In presence of CRTISO, the role of redox equivalents in dark isomerization of prolycopene, and the light induced signaling mechanisms for the regulation of carotenoid biosynthesis will be discussed.

Cardiac muscle contraction is based on the interaction between two filamentous systems: the thick filament, which is comprised of myosin and its accessory protein, cardiac myosin binding protein C (cMyBP-C) and the thin filament (TF), which is comprised of troponin (Tn), tropomyosin (Tpm), and filamentous actin (F-actin). Mutations in the gene encoding cMyBP-C are one of the most common causes of hypertrophic cardiomyopathy (HCM), a disease that affects approximately 1 in 500 people. The interaction between thick and thin filaments is regulated through translocation of Tpm cable by the Tncomplex in response to Ca 2þ followed by additional Tpm azimuthal movement upon binding of rigor myosin cross-bridges to F-actin. Therefore, activation of the TF is a two-step process which depends on both Ca 2þ -induced translocation of the Tpm and subsequent binding of rigor myosin heads. The N-terminal domain of cMyBP-C is comprised of three Ig-domains (C0, C1 and C2) and a regulatory linker (M-domain). We show that C1 Ig-domain is the only Igdomain that can activate the TFs at low Ca 2þ . 3D-reconstruction of frozen hydrated cardiac TFs decorated with C1 Ig-domain shows that the C1 Ig-domain on its own can translocate the Tpm cable on the surface of F-actin to the same extent as the combination of Ca 2þ and rigor myosin by tethering the Tpm cable to the subdomain-1 (SD1) of actin. Disruption of the interaction of the C1 Ig-domain interaction with either the SD1 of actin or Tpm cable by point mutations in C1 Ig-domain results in a complete loss of the C1-induced TF activation at low Ca 2þ . These data suggest how cMyBP-C can modulate the activation of the TF in heart muscle. . Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disorder stemming from several distinct mutations in the protein b-III-spectrin, including a leucine-to-proline missense mutation (L253P) in the actin-binding domain (ABD). This mutation causes a 1000-fold increase in affinity for actin and likely contributes to pathogenesis. However, the structural basis for the increase in affinity is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the b-III-spectrin ABD, harboring the L253P SCA5 mutation. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to an opening of the two CH domains (Avery et al., Nature Commun, 2017). This opening allows CH1 to bind actin aided by an unstructured N-terminal region that becomes a-helical upon binding. This N-terminal helix is crucial for association with actin, as its removal eliminates binding. In drosophila, the SCA-5 missense mutation results in aberrant localization of b-III-spectrin. Rather than being found throughout dendritic spines, b-III-spectrin is limited to the base of spine structures. This phenotype is consistent with the L253P mutation causing increased affinity for actin and, consequently, limiting b-III-spectrin's localization in neurons (Avery et al., Proc Natl Acad S...