bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dimethyl sulfoxide binding to globular proteins: A nuclear magnetic relaxation dispersion study
Authors: Jóhannesson Haukur , Denisov Vladimir P. , Halle Bertil 
Published: February 2009

AbstractThe 2H magnetic relaxation dispersion (NMRD) technique was used to characterize interactions of dimethyl sulfoxide (DMSO) with globular proteins. A difference NMRD experiment involving the N‐acetylglucosamine trisaccharide inhibitor, demonstrated that the DMSO 2H NMRD profile in lysozyme solution is due to a single DMSO molecule bound in the active cleft, with a molecular order parameter of 0.47 + 0.05 and a residence time in the range 10 ns to 5 ms. With the aid of transverse 2H relaxation data, the upper bound of the residence time was further reduced to 100 μs. A 1H shift titration experiment was also performed, yielding a binding constant of 2.3 + 0.3 M−1 at 27 °C. In contrast to lysozyme, no DMSO dispersion was observed for bovine pancreatic trypsin inhibitor (BPTI), indicating that a stable DMSO‐protein complex requires a cleft of appropriate geometry in addition to hydrogen‐bond and hydrophobic interactions.
Circular dichroism determination of class I MHC‐peptide equilibrium dissociation constants
Authors: Morgan Chantal S. , Holton James M. , Olafson Barry D. , Bjorkman Pamela J. , Mayo Stephen L. 
Published: February 2009

AbstractClass I major histocompatibility complex (MHC) molecules bind peptides derived from degraded proteins for display to T cells of the immune system. Peptides bind to MHC proteins with varying affinities, depending upon their sequence and length. We demonstrate that the thermal stability of the MHC‐peptide complex depends directly on peptide binding affinity. We use this correlation to develop a convenient method to determine peptide dissociation constants by measuring MHC‐peptide complex stability using thermal denaturation profiles monitored by circular dichroism.
Structural motifs at protein‐protein interfaces: Protein cores versus two‐state and three‐state model complexes
Authors: Tsai Chung‐Jung , Xu Dong , Nussinov Ruth 
Published: February 2009

AbstractThe general similarity in the forces governing protein folding and protein‐protein associations has led us to examine the similarity in the architectural motifs between the interfaces and the monomers. We have carried out extensive, all‐against‐all structural comparisons between the single‐chain protein structural dataset and the interface dataset, derived both from all protein‐protein complexes in the structural database and from interfaces generated via an automated crystal symmetry operation. We show that despite the absence of chain connections, the global features of the architectural motifs, present in monomers, recur in the interfaces, a reflection of the limited set of the folding patterns. However, although similarity has been observed, the details of the architectural motifs vary. In particular, the extent of the similarity correlates with the consideration of how the interface has been formed. Interfaces derived from two‐state model complexes, where the chains fold cooperatively, display a considerable similarity to architectures in protein cores, as judged by the quality of their geometric superposition. On the other hand, the three‐state model interfaces, representing binding of already folded molecules, manifest a larger variability and resemble the monomer architecture only in general outline. The origin of the difference between the monomers and the three‐state model interfaces can be understood in terms of the different nature of the folding and the binding that are involved. Whereas in the former all degrees of freedom are available to the backbone to maximize favorable interactions, in rigid body, three‐state model binding, only six degrees of freedom are allowed. Hence, residue or atom pair‐wise potentials derived from protein‐protein associations are expected to be less accurate, substantially increasing the number of computationally acceptable alternate binding modes (Finkelstein et al., 1995).
Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP‐dependent protein kinase
Authors: Gibson Robin M. , Ji‐Buechler Ying , Taylor Susan S. 
Published: February 2009

AbstractTwo classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP‐dependent protein kinase (cAPK), the heat‐stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Cα, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C‐subunit employs two distinct electrostatic surfaces to achieve high‐affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations, rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.
NMR chemical shift perturbation mapping of dna binding by a zinc‐finger domain from the yeast transcription factor ADR1
Authors: Schmiedeskamp Mia , Rajagopal Ponni , Klevit Rachel E. 
Published: February 2009

AbstractMutagenesis studies have revealed that the minimal DNA‐binding domain of the yeast transcription factor ADR1 consists of two Cys2‐His2 zinc fingers plus an additional 20 residues proximal and N‐terminal to the fingers. We have assigned NMR 1'H, 15N, and 13C chemical shifts for the entire minimal DNA‐binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single‐finger constructs, and 13Cα chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein‐DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N‐terminus of the finger 2 helix is affected. Furthermore, residues N‐terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein‐DNA interface. A striking correspondence is observed between the protein‐DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis.
Conformational studies of the N‐terminal lipid‐associating domain of human apolipoprotein C‐I by CD and 1H NMR spectroscopy
Authors: Rozek Annett , Buchko Garry W. , Kanda Patrick , Cushley Robert J. 
Published: February 2009

AbstractA peptide comprising the N‐terminal 38 residues of human apolipoprotein C‐I (apoC‐I(1‐38)) was synthesized using solid‐phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC‐I(1‐38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS‐bound apoC‐I(1‐38) was calculated from 464 NOE‐based distance restraints using distance geometry methods. ApoC‐I(1‐38) adopts a helical structure between residues V4 and K30 and an extended C‐terminus from Q31 when associated with SDS. The region K12‐G15 undergoes slow conformational exchange as indicated by above‐average amide resonance linewidths, large temperature coefficients, and fast exchange (<2 h) of backbone amide protons with deuterium. The mobility of K12‐G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC‐I(1‐38) is curved toward its hydrophobic face with bends of 125°, centered at K12/E13, and 150°, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC‐I(1‐38) and the previously obtained structure of the fragment apoC‐I(35‐53), we propose the secondary structure of intact apolipoprotein C‐I.
Solution structure of αtα, a helical hairpin peptide of de novo design
Authors: Fezoui Youcef , Connolly Peter J. , Osterhout John J. 
Published: February 2009

Abstractαtα is a 38‐residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui Y, Weaver DL, Osterhout JJ, 1995, Protein Sci 4286‐295). A previous study of the carboxylate form of αtα by CD and two‐dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675‐3679). Here, the solution structure of αtα as determined by two‐dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J‐couplings were used. One‐hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty‐three of the lowest‐energy structures consistent with the experimental restraints were analyzed. The results presented here show that αtα is comprised of two associating helices connected by a turn region.
Probing minimal independent folding units in dihydrofolate reductase by molecular dissection
Authors: Gegg Colin V. , Bowers Katherine E. , Matthews C. Robert 
Published: February 2009

AbstractMolecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven α‐helices or β‐strands and, in three cases, the adenine binding domain (residues 37‐86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1‐36 and 87‐159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1‐107 appears to have secondary and tertiary structure; however, spontaneous self‐association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37‐159 possesses significant secondary and tertiary structure. The urea‐induced unfolding of fragment 37‐159 in the presence of 0.5 M ammonium sulfate was found to be a well‐defined, two‐state process. The observation that fragment 37‐159 can adopt a stable native fold with unique, aromatic side‐chain packing is quite striking because residues 1‐36 form an integral part of the structural core of the full‐length protein.
Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability
Authors: Guddat Luke W. , Bardwell James C. A. , Glockshuber Rudi , Huber‐Wunderlich Martina , Zander Thomas , Martin Jennifer L. 
Published: February 2009

AbstractDsbA, a 21‐kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30‐Pro31‐His32‐Cys33). Unlike most protein disulfides, the active‐site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH.His32, one of the two residues between the active‐site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of ‐5‐7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild‐type protein.Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild‐type oxidized structure, including conservation of solvent atoms near the active‐site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.
pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pKa values
Authors: Singer Alex U. , Forman‐Kay Julie D. 
Published: February 2009

AbstractElectrostatic interactions in a complex of the phospholipase C‐γ, C‐terminal SH2 domain with a high‐affinity binding phosphopeptide representing the sequence around Tyr 1021 of the β platelet‐derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His βD4) and the phosphotyrosine (pTyrj phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pK, of His PD4 is also due to burial from solvent in a hydrogen‐bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa, values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C‐terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain‐phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.
Empirical parameterization of a model for predicting peptide helix/coil equilibrium populations
Authors: Andersen Niels H. , Tong Hui 
Published: February 2009

AbstractA modification of the Lifson‐Roig formulation of helix/coil transitions is presented; it (1) incorporates end‐capping and coulombic (salt bridges, hydrogen bonding, and side‐chain interactions with charged termini and the helix dipole) effects, (2) helix‐stabilizing hydrophobic clustering, (3) allows for different inherent termination probabilities of individual residues, and (4) differentiates helix elongation in the first versus subsequent turns of a helix. Each residue is characterized by six parameters governing helix formation. The formulation of the conditional probability of helix initiation and termination that we developed is essentially the same as one presented previously (Shalongo W, Stellwagen, E. 1995. Protein Sci 41161‐1166) and nearly the mathematical equivalent of the new capping formulation incorporated in the model presented by Rohl et al. (1996. Protein Sci 5:2623‐2637). Side‐chain/side‐chain interactions are, in most cases, incorporated as context dependent modifications of propagation rather than nucleation parameters. An alternative procedure for converting [θ]221 values to experimental fractional helicities (〈fH〉) is presented. Tests of the program predictions suggest this method may have some advantages both for designed peptides and for the analysis of secondary structure preferences that could drive the formation of molten‐globule intermediates on protein folding pathways. The model predicts the fractional helicity of 385 peptides with a root‐mean‐square deviation (RMSD) of 0.050 and locates (with precise definition of the termini in many cases) helices in proteins as well as competing methods. The propagation and nucleation parameters were derived from NMR data and from the CD data for a 79 peptide “learning set” for which an excellent fit resulted (RMSD = 0.0295). The current set of parameter corrections for capping boxes, helix dipole interactions, and side‐chain/side‐chain interactions (coulombic, hydrogen bonding, and hydrophobic clustering), although still under development, provide a significant improvement in both helix/coil equilibrium prediction for peptides and helix location in protein sequences. This is clearly evident in the rms deviations between CD measures and calculated values of fractional helicity for different classes of peptides before and after applying the corrections: for peptides lacking capping boxes and i/i + 3 and i/i + 4 side‐chain/side‐chain interactions RMSD = 0.044 (n = 164) versus RMSD = 0.054 (0.172 without the corrections, n = 221) for peptides that required context‐dependent corrections of the parameters. If we restrict the analysis to N‐acylated peptides with helix stabilizing side‐chain/side‐chain interactions (including N‐capping boxes), the degree to which our corrections account for the stabilizing interaction can be judged from the change in helicity underestimation, (〈fH〉calc ‐ 〈fH〈CD): ‐0.15 + 0.10, which is reduced to ‐0.018 + 0.048 (n = 191) upon applying the corrections.
Determination of pKa values of the histidine side chains of phosphatidylinositol‐specific phospholipase C from Bacillus cereus by NMR spectroscopy and site‐directed mutagenesis
Authors: Liu Tun , Ryan Margret , Dahlquist Frederick W. , Griffith O. Hayes 
Published: February 2009

AbstractTwo active site histidine residues have been implicated in the catalysis of phosphatidylinositol‐specific phospholipase C (PI‐PLC). In this report, we present the first study of the pKa values of histidines of a PI‐PLC. All six histidines of Bacillus cereus PI‐PLC were studied by 2D NMR spectroscopy and site‐directed mutagenesis. The protein was selectively labeled with 13Cϵ1‐histidine. A series of 1H‐13C HSQC NMR spectra were acquired over a pH range of 4.0‐9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H‐13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild‐type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.
Increased production of low molecular weight recombinant proteins in Escherichia coli
Authors: Belagaje Rama M. , Reams Stephen G. , Ly Stan C. , Prouty Walter F. 
Published: February 2009

AbstractA general method for obtaining high‐level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met‐Xaa‐protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N‐terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high‐level production of human insulin‐like growth factor‐1, human proinsulin, and their analogs. Furthermore, highly produced insulin‐like growth factor‐1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.
Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation
Authors: Dominici Paola , Moore Patrick S. , Castellani Silvia , Bertoldi Mariarita , Voltattorni Carla Borri 
Published: February 2009

AbstractCysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site‐directed mutagenesis. Compared to the wild‐type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kcat values of about 50% and 15%, respectively, at pH 6.8, while the Km values remain relatively unaltered for L‐3,4‐dihydroxyphenylalanine (L‐Dopa) and L‐5‐hydroxytryptophan (L‐5‐HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co‐enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys‐111→Ala mutant displays a reduced affinity for pyridoxal 5′‐phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D‐Dopa and the bound co‐enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild‐type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild‐type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 → 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys‐111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur J Biochem 201:393‐397). Moreover, our findings provide evidence that Cys‐111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active‐site conformation.
Crystallization and preliminary X‐ray crystallographic properties of Hsc20, a J‐motif co‐chaperone protein from Escherichia coli
Authors: Cupp‐Vickery Jill R. , Vickery Larry E. 
Published: February 2009

AbstractHsc20 is a 20‐kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X‐ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions α = 125.4 Å, b = 71.9 Å, c = 68.8 Å, and β = 97.0°. The crystals diffract to a minimum d‐spacing of ∼2.5 Å resolution, and a native data set was collected to 2.7 Å. The results of a self‐rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 Å3/Da and a solvent content of ∼53% in the crystals. Structure determination by isomorphous replacement is in progress.