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The role of calcium and other cations is characterized in the events of the catalytic turnover cycle. Certain 3d-cations bind to the active site of PLA and some are Ks cofactor for substrate or mimic binding, although without measurable kcat. The kcat cofactor role of a cation is seen in terms of a catalytic mechanism, where the water nucleophile in the expanded coordination area of the cation could be in a favorable near-attack conformation.

X-ray diffraction and infrared, Raman and nuclear magnetic resonance spectroscopic studies, as well as thermal gravimetric analysis were done to characterize the formation of a new class of layered, microcrystalline polymers from the hydrolysis of n-octadecyltrichlorosilane. The results revealed that the polymeric condensate is composed of periodic layers, each bearing a monomolecular layer of intercalated water, separated by crystalline assemblies of alkyl chains.

Human chorionic gonadotropin (hCG) maintains its biological activity through glycosylation of the alpha subunit. Glycosylation either makes hCG interact with a lectin-like part of the hCG receptor or reduces the affinity of the hormone for the hCG receptor, preventing its down-regulation. The glycan at Asn 52 plays an important role in the biological activity of hCG by extending into the surrounding solution irrespective of whether it is in the isolated alpha subunit or in complex with beta subunit. Conformations of the glycans do not differ greatly from that of free glycans in solution.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are the most versatile biocatalysts for synthesizing nonproteinogenic amino acids. α,α-Disubstituted quaternary amino acids, such as 1-aminocyclopentane-1-carboxylic acid (cycloleucine), are useful building blocks for pharmaceuticals. In this study, starting with the biosynthesis of fusarilin A, we discovered a family of PLP-dependent enzymes that can facilitate tandem carbon-carbon forming steps to catalyze an overall [3 + 2]-annulation. In the first step, the cycloleucine synthases use SAM as the latent electrophile and an in situ-generated enamine as the nucleophile for γ-substitution. Whereas previously characterized γ-replacement enzymes protonate the resulting α-carbon and release the acyclic amino acid, cycloleucine synthases can catalyze an additional, intramolecular aldol or Mannich reaction with the nucleophilic α-carbon to form the substituted cyclopentane. Overall, the net [3 + 2]-annulation reaction can lead to 2-hydroxy or 2-aminocycloleucine products. These studies further expand the biocatalytic scope of PLP-dependent enzymes.

G-protein coupled receptors (GPCRs) are the most prominent family of membrane proteins that serve as major targets for one-third of the drugs produced. A detailed understanding of the molecular mechanism of drug-induced activation and inhibition of GPCRs is crucial for the rational design of novel therapeutics. The binding of the neurotransmitter adrenaline to the β 2 -adrenergic receptor (β 2 AR) is known to induce a flight or fight cellular response, but much remains to be understood about binding-induced dynamical changes in β 2 AR and adrenaline. In this article, we examine the potential of mean force (PMF) for the unbinding of adrenaline from the orthosteric binding site of β 2 AR and the associated dynamics using umbrella sampling and molecular dynamics (MD) simulations. The calculated PMF reveals a global energy minimum, which corresponds to the crystal structure of β 2 AR-adrenaline complex, and a meta-stable state in which the adrenaline is moved slightly deeper into the binding pocket with a different orientation compared to that in the crystal structure. The orientational and conformational changes in adrenaline during the transition between these two states and the underlying driving forces of this transition are also explored. Based on the clustering of MD configurations and machine learning-based statistical analyses of time series of relevant collective variables, the structures and stabilizing interactions of these two states of the β 2 AR-adrenaline complex are also investigated.

Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental Streptomyces , pathogenic Mycobacterium employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and α-ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from Mycobacterium tuberculosis ( Mtb ), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in Mtb . A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between Streptomyces and Mycobacterium .

A highly stereoselective construction of 2,6-cis-disubstituted tetrahydropyrans was achieved by using an intramolecular amide enolate alkylation with KHMDS. The efficiency and practicality of this methodology was successfully demonstrated in the total synthesis of (-)-centrolobine (1).

In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.

13C relaxation studies on side-chain methyl groups in proteins typically involve measurements on (13)CHD(2) isotopomers, where the (13)C relaxation mechanism is particularly straightforward in the presence of a single proton. While such isotopomers can be obtained in proteins overexpressed in bacteria by use of (13)C enriched and fractionally deuterated media, invariably all possible (2)H isotopomers are obtained. This results in a loss of both resolution and sensitivity, which becomes particularly severe for larger proteins. We describe an approach that overcomes this problem by chemical synthesis of amino acids containing a pure (13)CHD(2) isotopomer. We illustrate the benefits of this approach in (13)C side-chain relaxation measurements on the mouse major urinary protein selectively enriched with [gamma(1),gamma(2)-(13)C(2),alpha,beta,gamma(1),gamma(1),gamma(2),gamma(2)-(2)H(6)] valine. Relaxation measurements in the absence and presence of pyrazine-derived ligands suggest that valine side-chain dynamics do not contribute significantly to binding entropy.

Equilibrium pressures for the dissociation of carbon dioxide hydrates confined in silica gel pores of nominal radii 7.5, 5.0, and 3.0 nm were measured over a wide temperature range and were observed to be higher than those for bulk carbon dioxide hydrate. Models that have been previously reported in the literature are used to determine the pore radius involved in each equilibrium associated with these data, exactly reproducing the experimental equilibrium pressure. Based on these models, pore volume distributions are reconstructed and compared to those obtained from nitrogen desorption isotherms. This comparison indicates that in the nominal 7.5 nm pores the hydrate formed nearly uniformly in the available pores, while in the nominal 5.0 and 3.0 nm pores it did not.

The conformational properties in solution of the glycans on the alpha subunit of recombinant human chorionic gonadotropin are described, using high-resolution multinuclear NMR studies on uniformly 13C, 15N-enriched recombinant glycoprotein expressed in CHO cells. The glycan important for full biological activity of hCG, namely, that at Asn 52, appears to extend into solution both in the isolated alpha subunit and in complex with the beta subunit. The disposition of this glycan with respect to the protein backbone suggests that glycosylation maintains full biological activity of hCG either by interacting with a lectin-like region of the hCG receptor or by reducing the affinity of the hormone for the hCG receptor and preventing its down-regulation.