Energetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses

We reported previously the effects of both osmotic and curvature stress on fusion between poly(ethylene glycol)-aggregated vesicles. In this article, we analyze the energetics of fusion of vesicles of different curvature, paying particular attention to the effects of osmotic stress on small, highly curved vesicles of 26 nm diameter, composed of lipids with negative intrinsic curvature. Our calculations show that high positive curvature of the outer monolayer "charges" these vesicles with excess bending energy, which then releases during stalk expansion (increase of the stalk radius, r(s)) and thus "drives" fusion. Calculations based on the known mechanical properties of lipid assemblies suggest that the free energy of "void" formation as well as membrane-bending free energy dominate the evolution of a stalk to an extended transmembrane contact. The free-energy profile of stalk expansion (free energy versus r(s)) clearly shows the presence of two metastable intermediates (intermediate 1 at r(s) approximately 0 - 1.0 nm and intermediate 2 at r(s) approximately 2.5 - 3.0 nm). Applying osmotic gradients of +/-5 atm, when assuming a fixed trans-bilayer lipid mass distribution, did not significantly change the free-energy profile. However, inclusion in the model of an additional degree of freedom, the ability of lipids to move into and out of the "void", made the free-energy profile strongly dependent on the osmotic gradient. Vesicle expansion increased the energy barrier between intermediates by approximately 4 kT and the absolute value of the barrier by approximately 7 kT, whereas compression decreased it by nearly the same extent. Since these calculations, which are based on the stalk hypothesis, correctly predict the effects of both membrane curvature and osmotic stress, they support the stalk hypothesis for the mechanism of membrane fusion and suggest that both forms of stress alter the final stages, rather than the initial step, of the fusion process, as previously suggested.     

A Crosstalk between the Biorhythms and Gatekeepers of Longevity: Dual Role of Glycogen Synthase Kinase-3

Biochemistry (Moscow) - This review discusses genetic and molecular pathways that link circadian timing with metabolism, resulting in the emergence of positive and negative regulatory feedback...     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Arrestins: structural disorder creates rich functionality

Arrestins are soluble relatively small 44–46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstrate preference for each of the known arrestin conformations: free, receptor-bound, and microtubule-bound. Recent evidence suggests that conformational flexibility in every functional state is the defining characteristic of arrestins. Flexibility, or plasticity, of proteins is often described as structural disorder, in contrast to the fixed conformational order observed in high-resolution crystal structures. However, protein-protein interactions often involve highly flexible elements that can assume many distinct conformations upon binding to different partners. Existing evidence suggests that arrestins are no exception to this rule: their flexibility is necessary for functional versatility. The data on arrestins and many other multi-functional proteins indicate that in many cases, “order” might be artificially imposed by highly non-physiological crystallization conditions and/or crystal packing forces. In contrast, conformational flexibility (and its extreme case, intrinsic disorder) is a more natural state of proteins, representing true biological order that underlies their physiologically relevant functions.     

A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on L-arginine hydroxylation and GN-hydroxy-L-arginine oxidation

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of L-arginine (Arg) with formation of an intermediate, GN-hydroxy-L-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H(4)B and instability of the heme-CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H(4)B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 microM NHA and 0.39 microM H(4)B to reach the half-maximal NHA-driven NO formation rate (EC(50)), while R365L needed 59 microM NHA and 0.73 microM H(4)B to achieve EC(50). The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.