Cosolutes Modify Alkaline Phosphatase Catalysis through Osmotic Stress and Crowding Mechanisms.

Examining the effects of different cosolutes on enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, milieu. Viewing cosolute 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, , caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme-substrate complex formation.

Intrinsically disordered N-terminus of calponin homology-associated smooth muscle protein (CHASM) interacts with the calponin homology domain to enable tropomyosin binding.

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.

Proteome of the Caenorhabditis elegans oocyte.

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.

Structural mass spectrometry of the alpha beta-tubulin dimer supports a revised model of microtubule assembly.

The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of alphabeta-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.

A unique mode of microtubule stabilization induced by peloruside A.

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.

Quantitating the statistical distribution of deuterium incorporation to extend the utility of H/D exchange MS data.

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.

Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts.

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.