No abstract

Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

The aggregates of α-synuclein bear a close connection with Parkinson’s disease, which is largely characterized by the loss of the dopaminergic neurons. Dopamine promotes the formation of undesirable sodium dodecyl sulfate (SDS)-resistant oligomers of α-synuclein. In this study, we have shown that the inhibition of fibrillation by an additive may not always be the ultimate deciding factor in the context of its potential as a successful additive. Copper promotes the fibrillation of α-synuclein in buffer alone but inhibits the formation of SDS-resistant oligomers in the presence of dopamine. Glycerol, on the other hand, increases the population of such dopamine-mediated SDS-resistant oligomers. We speculate such an effect to be a manifestation of the distinct oxidation pathway of dopamine in the presence of copper.

Higher amounts of phorbol esters are present in oil than cake but bio-diesel and glycerol are free of phorbol esters. The other anti-nutritional components such as trypsin inhibitors, phytates and curcin are present in cake, so the cake should be detoxified before being used for animal feed.

Background The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG‐GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. Objective Here, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. Methods CDGI interaction with phosphoinositides was studied by lipid co‐sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. Results Lipid co‐sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)‐biphosphate (PIP2) and phosphatidylinositol (3,4,5)‐triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co‐sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. Conclusion Our studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)‐biphosphate and phosphatidylinositol (3,4,5)‐triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.

The ability of cells and tissues to differentially resist or adapt to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding are unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of these variants into Vcn biosensors did not perturb Vcn conformation, but reduced Vcn loading consistent with loss of directional catch bonding. Expression of Vcn variants perturbed the coalignment of FAs and F-actin and directed cell migration, establishing key cellular functions for Vcn directional catch bonding.

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.