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PubMed2002

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.

Chemical Precipitation↗
PubMed2002

Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase.

The motions of key residues at the substrate binding site of lactate dehydrogenase (LDH) were probed on the 10 ns to 10 ms time scale using laser-induced temperature-jump relaxation spectroscopy employing both UV fluorescence and isotope-edited IR absorption spectroscopy as structural probes. The dynamics of the mobile loop, which closes over the active site and is important for catalysis and binding, were characterized by studies of the inhibitor oxamate binding to the LDH/NADH binary complex monitoring the changes in emission of bound NADH. The bound NAD-pyruvate adduct, whose pyruvate moiety likely interacts with the same residues that interact with pyruvate in its ternary complex with LDH, served as a probe for any relative motions of active site residues against the substrate. The frequencies of its C=O stretch and -COO(-) antisymmetric stretch shift substantially should any relative motion of the polar moieties at the active site (His-195, Asp-168, Arg-109, and Arg-171) occur. The dynamics associated with loop closure are observed to involve several steps with motions from 1 to 300 microms. Apart from the "melting" of a few residues on the protein's surface, no kinetics were observed on any time scale in experiments of the bound NAD-pyr adduct although the measurements were made with a high degree of accuracy, even for final temperatures close to the unfolding transition of the protein. This is contrary to simple physical considerations and models. These results show that, once a productive protein/substrate complex is formed, the binding pocket is very rigid with very little, if any, motion apart from the mobile loop. The results also show that loop opening involves concomitant movement of the substrate out of the binding pocket.

Catalysis↗
PubMed2005

Photoaffinity labeling with 8-azidoadenosine and its derivatives: chemistry of closed and opened adenosine diazaquinodimethanes.

The reactive intermediate produced upon photolysis of 8-azidoadenosine was studied by chemical trapping studies, laser flash photolysis with UV-vis and IR detection, and modern computational chemistry. It is concluded that photolysis of 8-azidoadenosine in aqueous solution releases the corresponding singlet nitrene which rapidly tautomerizes to form a closed adenosine diazaquinodimethane in less than 400 fs. A perbenzoylated derivative of 8-azidoadenosine cannot undergo this tautomerization, and instead, it fragments upon photolysis to form an opened adenosine diazaquinodimethane. The singlet nitrene is too short-lived to be observed and, thus, to relax to the lowest triplet state or to become covalently attached to targeted biological macromolecules. The pivotal closed adenosine diazaquinodimethane, the product of nitrene tautomerization, has a lifetime of ca. 1 min or longer in water and in HEPES buffer at ambient temperature. However, this intermediate reacts rapidly with good nucleophiles such as amines, thiols, and phenolates, and significantly more slowly with weak nucleophiles such as alcohols and water. On the basis of these studies, it is clear that the closed adenosine diazaquinodimethane, and not the singlet or triplet nitrene, is the pivotal reactive intermediate involved in photolabeling and cross-linking studies using the 8-azidoadenosine family of photoaffinity labeling reagents.

Adenosine↗
PubMed1996

Reduction of cyclic and acyclic diazene derivates by Azotobacter vinelandii nitrogenase: diazirine and trans-dimethyldiazene.

Nitrogenase reduces N2 to NH3, but the mechanistic details are unclear. Diazene (N2H2), a proposed 2e-/2H+ intermediate on the reduction pathway, is labile under typical enzyme assay conditions, and no firm evidence is available on whether or not it can be reduced by or inhibit nitrogenase. In this paper, we compare the interactions of Azotobacter vinelandii (Av) nitrogenase with two diazene analogues: diazirine, a photolabile diazene containing the azo (-N=N-) group in a strained, three-membered ring, and trans-dimethyldiazene, a diazene containing an unstrained trans-disubstituted N=N bond. Diazirine is reduced by nitrogenase under specific conditions to methane, methylamine, and ammonia in a ratio of ca. 1:2:4-5 with a Km value for all three products similar (0.05-0.09 mM) to that of dinitrogen (0.06-0.12 mM). The Km value of diazirine does not depend on the ratio of nitrogenase Fe protein (Av2) to nitrogenase MoFe protein (Av1) at Av2:Av1 ratios of 0.71 and 14.9. Diazirine potently and competitively inhibits acetylene reduction by Av nitrogenase with Ki = 0.03 mM and is predicted to inhibit H2 evolution completely at pressures >> Km. The experimental Henry's Law constant (1.50 M/atm) determined for trans-dimethyldiazene in H2O shows that it has about 20-fold higher solubility than diazirine in water at 30 degrees C. trans-Dimethyldiazene is reduced by nitrogenase under specific conditions to ammonia, methane and methylamine in a ratio of ca. 1:1:1 with Km values for the three products of 0.51-0.58 M. The product ratio does not change significantly when the component ratio (Av2:Av1 ) is varied over 2.06-13.62. trans-Dimethyldiazene reduction is inhibited noncompetitively by CO and C2H2 with Ki values of ca. 0.0008 and 0.006 atm, respectively. The results are discussed with respect to the stereoelectronic differences between the two azo substrates. A "random-edge" reduction is compared with alternative schemes for the diazirine reduction. For trans-dimethyldiazene, initial C-N cleavage is proposed to yield CH4 and a bound CH3N2H species, which is then reduced to CH3NH2 and NH3.

Acetylene↗
PubMed2000

Protein thioacylation. 1. Reagents design and synthesis.

Thioacylation is a new way for protein chemical modification. Carboxylic dithioesters and -acids react selectively and rapidly at room temperature with aliphatic amines such as lysine epsilon-amino groups leading to thioamide formation, without any other reagent or catalyst. Various thioacylating reagents were synthesized: monofunctional dithioesters bearing on the acylating end various chemical groups such as: aliphatic chains, phenyl group, mono- and dicarboxylic acids, dialkylphosphonic ester, phosphonic acid, thiol, phenol, or quaternary ammonium group. Bifunctional dithioesters containing either a polymethylene chain or an ethylene oxide oligomer as spacer group as well as some mono- and bis(dithio acids) are described. Applications of thioacylation may be involved either in enzyme chemical modifications or in the obtention of new materials from proteins. Bifunctional reagents might be used as cross-linking or coupling reagents.

Acylation↗