Examining the effects of different cosolutes on in vitro enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, in vivo milieu. Viewing cosolute in vitro influences on a model enzyme, calf intestinal alkaline phosphatase, as a combination of competitive and uncompetitive behaviors provided quantitative insights into their effects on catalysis. Observed decreases in the apparent specificity constant, Kasp, caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme-substrate complex formation. This competitive inhibition appeared to be driven by osmotic stress. Dextran 6 K and sucrose strongly impeded the subsequent conversion of the bound substrate into a free product, which was marked by sharp reductions in V-max, uncompetitive inhibition. For the same step, smaller noncarbohydrate cosolutes, triethylene glycol, polyethylene glycol 400, and betaine, also behaved as uncompetitive inhibitors but to a lesser extent. However, polyethylene glycol 8000 and 20,000 were uncompetitive activators, increasing V-max. Polyethylene glycol of molecular weight 1000 displayed intermediate effects between these two groups of noncarbohydrate cosolutes. These results suggested that crowding has a strong influence on free product formation. The combination of competitive and uncompetitive effects and mixed behaviors, caused by the cosolutes on calf intestinal alkaline phosphatase kinetics, was consistent with the trends seen in similar enzyme-cosolute studies. It is proposed that the double-displacement mechanism of alkaline phosphatases, shared by many other enzymes, could be the root of this general observation.

Many enzymes have evolved to function in aqueous environments filled with many other solutes. In addition to specific interactions, these co-solutes may also indirectly modify enzyme behaviour through various means generally referred to as crowding. Using UV-visible spectrometry, we have measured the kinetics of para-nitrophenol phosphate (PNPP) hydrolysis catalyzed by bovine intestinal alkaline phosphatases in the presence of various solutes such as betaine, sucrose, triethylene glycol and polyethylene glycols. The effects of these solutes were quantitated using the “classical” small-molecule language of competitive and uncompetitive inhibition. Nearly all the solutes increased the difficulty of enzyme/substrate formation. In other words, they acted as competitive inhibitors. But at the same time, these same solutes showed a variety of uncompetitive effects ranging from a significant decrease in the rate of product formation rate using sucrose, to noticeable increases in the same product formation rate with polyethylene glycol 1000 and 8000 molecular weight. Using the classical language of small-molecule inhibition provides new perspectives on the effects of crowding on enzyme kinetics.

Reverse micelles are formed by the aggregation of water inside of a bilayer formed by a surfactant in a non-polar solvent. The nanometer scale pool of water within the reverse micelles provides an environment to study the effects of confinement on enzymes as measured by changes in kinetics. The size of these reverse micelles is directly related to the ratio of water and surfactant. We and others have been studying the effects of crowding, the presence of large concentrations of solutes, on enzyme kinetics. What effect would it have on enzyme kinetics if these solutes were added to the confined environment of the reverse micelle? We have studied the effects of confinement on alpha-chymotrypsin in a system using dioctyl sulfosuccinate (AOT) as a surfactant, isooctane as the non-polar solvent, and N-succinyl-L-phenylalanine p-nitroanilide (SPN) as the substrate. Results will be analyzed within the Michaelis-Menten framework. * Indicates faculty mentor.

Enzymes realize their form and function in an aqueous environment filled with many other solutes. In addition to specific interactions, these co-solutes may also indirectly modify enzyme behavior through changes in osmotic pressure (i.e. the chemical potential of water). Using UV-visible spectrometry, we have measured the kinetics of bovine intestinal alkaline phosphatase catalyzed para-nitrophenol phosphate hydrolysis in the presence of various neutral solutes such as betaine, sucrose, triethylene glycol and polyethylene glycols. When analyzed using Lineweaver-Burk plots, the inhibition mechanism of these osmolytes appear analogous to "regular" small-molecule, mixed enzyme inhibitors (see figure). This suggests that these osmolytes can affect the two classic Michaelis-Menten "steps": substrate (S) binding to enzyme (E) (i.e. E+S ↔ ES), competitive inhibition, and the subsequent conversion of substrate to product (P) (i.e. ES → E+P), uncompetitive inhibition. These observed effects could be interpreted as different degrees of osmolyte exclusion from regions on alkaline phosphatase critical to these steps. Furthermore, the amount and location of this exclusion would be sensitive to the steric and chemical differences between these solutes. These results highlight the importance taking into consideration the actually complex environment in which enzymes operate.

The calponin homology-associated smooth muscle (CHASM) protein plays an important adaptive role in smooth and skeletal muscle contraction. CHASM is associated with increased muscle contractility and can be localized to the contractile thin filament via its binding interaction with tropomyosin. We sought to define the structural basis for the interaction of CHASM with smooth muscle tropomyosin as a first step to understanding the contribution of CHASM to the contractile capacity of smooth muscle. Herein, we provide a structure-based model for the tropomyosin-binding domain of CHASM using a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and NMR analyses. Our studies provide evidence that a portion of the N-terminal intrinsically disordered region forms intramolecular contacts with the globular C-terminal calponin homology (CH) domain. Ultimately, cooperativeness between these structurally dissimilar regions is required for CHASM binding to smooth muscle tropomyosin. Furthermore, it appears that the type-2 CH domain of CHASM is required for tropomyosin binding and presents a novel function for this protein domain.

Oocytes were purified from the temperature-sensitive fertilization-defective fer-1(b232ts) mutant of the nematode Caenorhabditis elegans and used for comprehensive mass spectrometric analysis. Using stringent criteria, 1165 C. elegans proteins were identified; at lower stringency, an additional 288 proteins were identified. We validate the high degree of sample purity and evaluate several possible sources of bias in the proteomic data. We compare the classes of proteins identified in the current oocyte proteome with protein classes identified in our previously determined oocyte transcriptome. The oocyte proteome appears enriched in proteins likely to be needed immediately upon fertilization, whereas the transcriptome appears enriched in molecules and processes needed later in embryogenesis. The current study provides fundamental background information for future more detailed studies of oocyte biology.

The smoothelin‐like 1 protein (SMTNL1) modulates muscle contractile activity, particularly in exercise adaptation. SMTNL1 bears sequence similarity to smoothelin, a smooth muscle differentiation marker, particularly in its calponin homology (CH) domain. The interaction of SMTNL1 with tropomyosin (TM) is dependent on the CH domain and some portion of a central, unfolded region (aa 197–342) of SMTNL1. These data are based on pull‐down studies with SMTNL1 truncations, and reinforced by isothermal titration calorimetry experiments. We hypothesize that independent sections of the SMTNL1 protein cooperate to form a functional TM binding surface. We will elucidate the specific surfaces of SMTNL1 involved in TM binding using hydrogen‐deuterium exchange mass spectroscopy (HXMS). To this end, we have mapped pepsin‐derived mass fragments covering 94% of the SMTNL1‐CH domain, and 83% of a functional TM‐binding truncate (SMTNL1‐∆N, aa 196–459). These maps will be compared with deuterated SMTNL1 samples, and with SMTNL1 samples deuterated while bound to TM. The deuterium incorporated is calculated for each SMTNL1 peptide to reveal the interaction interface. The objectives will address the molecular and structural elements of the SMTNL1‐TM interaction and provide a framework for the physiological role of SMTNL1 in vascular smooth muscle biology. The research was supported by HSFC and CIHR.

The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of alpha beta-tubulin. While this has been appreciated for many years, there is ongoing debate over the molecular basis of this instability and the possible role of altered nucleotide occupancy in the induction of a conformational change in tubulin. The debate has organized around seemingly contradictory models. The allosteric model invokes nucleotide-dependent states of curvature in the free tubulin dimer, such that hydrolysis leads to pronounced bending and thus disruption of the lattice. The more recent lattice model describes a predominant role for the lattice in straightening free dimers that are curved regardless of their nucleotide state. In this model, lattice-bound GTP-tubulin provides the necessary force to straighten an incoming dimer. Interestingly, there is evidence for both models. The enduring nature of this debate stems from a lack of high-resolution data on the free dimer. In this study, we have prepared alpha beta-tubulin samples at high dilution and characterized the nature of nucleotide-Induced conformational stability using bottom-up hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation experiments. These experiments were accompanied by molecular dynamics simulations of the free dimer. We demonstrate an intermediate state unique to GDP-tubulin, suggestive of the curved colchicine-stabilized structure at the intradimer interface but show that intradimer flexibility is an important property of the free dimer regardless of nucleotide occupancy. Our results indicate that the assembly properties of the free dimer may be better described on the basis of this flexibility. A blended model of assembly emerges in which free-dimer allosteric effects retain importance, in an assembly process dominated by lattice-induced effects.

Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the alpha/beta-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of beta-tubulin at a previously unknown ligand site, rather than on alpha-tubulin as suggested in earlier studies.

The distance dependence for the preferential exclusion of several salts and neutral solutes from hydroxypropyl cellulose (HPC) has been measured via the effect of these small molecules on the thermodynamic forces between HPC polymers in ordered arrays. The concentration of salts and neutral solutes decreases exponentially as the spacing between apposing nonpolar HPC surfaces decreases. For all solutes, the spatial decay lengths of this exclusion are remarkably similar to those observed between many macromolecules at close spacings where intermolecular forces have been ascribed to the energetics of water structuring. Exclusion magnitudes depend strongly on the nature and size of the particular salt or solute; for the three potassium salts studied, exclusion follows the anionic Hofmeister series. The change in the number of excess waters associated with HPC polymers is independent of solute concentration suggesting that the dominating interactions are between solutes and the hydrated polymer. These findings further confirm the importance of solvation interactions and reveal an unexpected unity of Hofmeister effects, preferential hydration, and hydration forces.

Measuring the statistical distribution of deuterium incorporated into enzymatically derived peptide fragments provides a valuable dimension to hydrogen/deuterium exchange mass spectrometry data. In this paper, we will discuss our improvement to the linear least-squares method for determining this distribution, through the addition of "zeroes" to the end of the deuterated isotopic envelope, to partially compensate for data truncation due to finite instrumental signal-to-noise ratios. The value of the distribution is demonstrated in a simple experimental example, where the linearity between average deuteration and percent D2O used to label test peptides hides a more complex relationship between the site-labeling probability and the total number of sites. This method offers the opportunity to resolve cases where a single peptide experiences distinct, independent biochemical states with each bearing a unique average deuteration; this can occur when a protein is modified to substoichiometric levels. From the experimentally determined distribution of a heterogeneously deuterated peptide, it was possible to extract the average deuteration of each component of the mixture.

Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.

Packing free energies and structural transitions of concentrated arrays of guar galacto-mannan macromolecules and of guars modified by hydroxypropyl substitution (HPG) have been studied using the osmotic stress method combined with X-ray scattering. All show a liquid crystalline structure with packing free energies that are very similar for guar and HPG and well described by the model of Selinger and Bruinsma for entropic steric repulsion between chains. In addition, a transition from the liquid crystalline form to a crystalline structure is observed as native guar becomes more densely packed. This transition is related to the propensity of guar to form intermolecular hydrogen bonds in solutions. Hydroxypropyl substitution of galactomannan hydroxyl groups causes steric interference that decreases the stability of this hydrogen-bonded crystalline structure. Even for moderately hydroxypropyl-substituted guar (similar to0.3 HP/sugar residue), the transition occurs at a much higher osmotic pressure than for native guar. The extra work needed to crystallize this HPG compared with guar is calculated to be 3 kT/mannose unit or 6-7 kT per hydroxypropyl group. No transition was found for more highly substituted guars. Urea increased the osmotic pressure necessary for the transition of guar but also resulted in new crystalline packing structure.

Contrary to the accurate, hard-sphere depiction of monomeric hemoglobin in solution, sickle cell hemoglobin (HbS) polymerization/gelation requires attention to molecular interactions. From the temperature dependence of the osmotic compressibility of HbS gels, we were able to extract the entropy increase for concentrating HbS in this phase. Normalized per mole of water removed, the entropy increase from gel compression DeltaS(gel) is four times the previously measured DeltaS(trans), for the transition from monomeric HbS solution to HbS gel. The positive entropy change cannot emerge from the assembly of hard spheres but can indicate remodeling of HbS fibers driven by release of ordered water. The fourfold difference in DeltaS(gel) and DeltaS(trans) suggests that the act of initial fiber/gel formation from monomeric solution differs from the process of further polymerization due to tighter packing within the gel phase.

In this paper, we describe a low power system using Polycapillary collimating and focusing optics that were designed to collect Cu Ka radiation from an Oxford Ultra-Bright micro-focus source for X-ray powder diffraction measurements. The characterizations of the source and polycapillary optics are presented. A collimator with two apertures was used to block high energy X-rays. An optic alignment system was designed to optimize coupling between the optics and the source, taking into account the maximum radiation direction from the source. Several powder sample data sets were collected with this system and their qualities are compared with data sets from the same samples taken with an Enraf-Nonius FR590 sealed-tube source system. Discussion is also presented for further improving the performance of this low power system.

The structure of an “open state” of crystalline profilin:β-actin has been solved to 2.65 Å by X-ray crystallography. The open-state crystals, in 1.8 M potassium phosphate, have an expanded unit cell dimension in thecdirection of 185.7 Å compared with 171.9 Å in the previously solved ammonium sulphate-stabilized “tight-state” structure. The unit cell change between the open and the tight states is accompanied by large subdomain movements in actin. Furthermore, the nucleotide in the open state is significantly more exposed to solvent, and local conformational changes in the hydrophobic pocket surrounding cysteine 374 occur during the transition to the tight state. Significant changes were observed at the N terminus and in the DNase-I binding loop. Neither the structure of profilin nor its contact with β-actin are affected by the changes in the unit cell. Applying osmotic pressure to profilin:β-actin crystals brings about a collapse of the unit cell comparable with that seen in the open to tight-state transition, enabling an estimate of the work required to cause this transformation of β-actin in the crystals. The slight difference in energy between the open and collapsed states explains the extreme sensitivity of profilin:β-actin crystals to changes in chemical and thermal environment.