Terrence A. Scahill

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

SummaryHuman ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitins role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly 15N-enriched ubiquitin. Sequence-specific 1H and 15N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets. Because of 1H chemical shift degeneracies, the increased resolution provided by the 15N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of β-sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an α-helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of β-sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of 1Hα chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional 1H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].

Reaction of aryl chromium carbene complexes with ethyl propiolate a versatile vinyl ether formation

Abstract Arylmethoxychromium-carbene complexes reacted with ethyl propiolate in the presence of alcohol to form and yields of aryl vinyl ether derivatives of malonate.

Reaction of a pyrrole-carbene chromium complex with alkynes: a facile hydroindoloquinone formation with in-situ protection.

Abstract Cycloaddition reaction of a pyrrole-carbene chromium complex with alkyne in the presence of acetic anhydride and triethylamine provided the acetylated hydroindoloquinone derivative.

Reactions of aryl chromium carbene complexes with alkoxalkyne: O-Quinonemethide formation and unusual diels-alder dimerization

Abstract Reactions of a pyrrole- and a phenyl-carbene chromium complexes with 3-alkoxy-1-ethoxy-1-butyne produced dimers through o-quinone-methide formation from the alkyne-carbene cycloaddition products and subsequent Diels-Alder dimerization.

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

SummarySequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H−15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNHα coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel β-sheet and four α-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.

Furans in synthesis 101. An efficient construction of the bicyclo[5.3.0]decane ring system of fastigilin-C

Abstract The efficient preparation of an advanced intermediate for the synthesis of Fastigilin-C 2 , via a furan terminated cationic cyclization, is described.

Solution structure of human interleukin-1 receptor antagonist protein.

Interleukin‐1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin‐1 receptor. In contrast to IL‐1β IRAP binds to the IL‐1 receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153‐residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL‐1 receptor binding are conserved between IRAP and IL‐1β. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.

An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: Implications for drug transport

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R1–D‐Xaa–D‐Phe–NHMe) homologues were each dissolved in a micellar d38‐dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen‐bond potential, and charge were either tightly controlled or well‐characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide‐NH–H2O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with 3JHH‐derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that—other factors being equal—flexible dipeptides having a propensity to fold together nonpolar N‐ and C‐terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.

Purification and structural characterization of the CD11b/CD18 integrin α subunit I domain reveals a folded conformation in solution

The α subunits of the leukocyte CD11/CD18 integrins contain a ∼200 amino acid ‘inserted’ or I domain. The I domain of the cell‐surface Mac‐1(CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM‐1. The I domain from the Mac‐1 α subunit has been expressed in Escherichia coli as a soluble GST‐fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the α subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.

Proton, carbon, and nitrogen chemical shifts accurately delineate differences and similarities in secondary structure between the homologous proteins IRAP and IL-1β

Summary1Hα,13Cα, and15Nα secondary chemical shifts, defined as the difference between the observed value and the random coil value, have been calculated for interleukin-1 receptor antagonist protein and interleukin-1β. Averaging of the secondary chemical shifts with those of adjacent residues was used to smooth out local effects and to obtain a correlation dependent on secondary structure. Differences and similarities in the placement of secondary structure elements in the primary segdences of these structurally homologous proteins are manifested in the smoothed secondary chemical shifts of all three types of nuclei. The close correlation observed between the secondary chemical shifts and the previously defined locations of secondary structure, as defined by traditional methods, exemplifies the advantage of chemical shifts to delineate regions of secondary structure.