bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from antarctic cod
Authors: Lane Andrew N. , Hays Lisa M. , Crowe Lois M. , Crowe John H. , Feeney Robert E. 
Published: February 2009

AbstractThe 1H and 13C NMR spectra of a 14‐residue antifreeze glycopeptide from Antarctic cod (Tetramatornus horchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 °C of 3‐4 ns. The relaxation data and lack of long‐range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16‐residue analogue containing an Arg‐Ala dipeptide added to the C‐terminus. Molecular modeling also showed no evidence of long‐range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or β turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.
Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Authors: Sun Dunming , Jessen Sven , Liu Chunhui , Liu Xiuping , Najmudin Shabir , Georgiadis Millie M. 
Published: February 2009

AbstractReverse transcriptase is an essential retroviral enzyme that uses RNA‐ and DNA‐directed DNA polymerase activities as well as an RNaseH activity to synthesize a double‐stranded DNA copy of the single‐stranded RNA genome. In an effort to obtain high‐resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment‐nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose‐binding protein
Authors: Center Rob J. , Kobe Bostjan , Wilson Kirilee A. , Teh Trazel , Kemp Bruce E. , Poumbourios Pantelis , Howlett Geoffrey J. 
Published: February 2009

AbstractWe present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type I (HTLV‐1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C‐terminal α‐helix to the predicted N‐terminal α‐helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X‐ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N‐terminal α‐helical sequences.
Role of P225 and the C136‐C201 disulfide bond in tissue plasminogen activator
Authors: Vindigni Alessandro , Cera Enrico Di 
Published: February 2009

AbstractThe protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor‐1 (PAI‐1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at PI, P2, and P3 reveals a strong preference for Arg over Lys at PI, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+was discovered. The divalent cation presumably binds to H188 near Dl89 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 μM. In an attempt to engineer Na+binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+‐dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100‐fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI‐I and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+to inhibit the enzyme. Elimination of the C136‐C201 disulfide bond, that is absent in all Na+‐dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+‐dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+binding site of allosteric serine proteases.
Molecular mechanisms of resistance: Free energy calculations of mutation effects on inhibitor binding to HIV‐1 protease
Authors: Rick Steven W. , Topol Igor A. , Burt Stanley K. , Erickson John W. 
Published: February 2009

AbstractThe changes in the inhibitor binding constants due to the mutation of isoleucine to valine at position 84 of HIV‐1 protease are calculated using molecular dynamics simulations. The calculations are done for three potent inhibitors‐KNI‐272, L‐735,524 (indinavir or MK‐639), and Ro 31‐8959 (saquinavir). The calculations agree with the experimental data both in terms of an overall trend and in the magnitude of the resulting free energy change. HIV‐1 protease is a homodimer, so each mutation causes two changes in the enzyme. The decrease in the binding free energy from each mutated side chain differs among the three inhibitors and correlates well with the size of the cavities induced in the protein interior near the mutated residue. The cavities are created as a result of a mutation to a smaller side chain, but the cavities are less than would be predicted from the wild‐type structures, indicating that there is significant relaxation to partially fill the cavities.
Enzyme‐mononucleotide interactions: Three different folds share common structural elements for atp recognition
Authors: Denessiouk Konstantin A. , Lehtonen Jukka V. , Johnson Mark S. 
Published: February 2009

AbstractThree ATP‐dependent enzymes with different folds, cAMP‐dependent protein kinase, D‐Ala:D‐Ala ligase and the α‐subunit of the α2β2 ribonucleotide reductase, have a similar organization of their ATP‐binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their β‐sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the “AMP” moieties of their ATP‐ligands. One lysine residue conserved throughout the three families binds the alpha‐phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP‐binding by three unrelated proteins.
The magnitude of changes in guanidine‐HC1 unfolding m‐values in the protein, iso‐1‐cytochrome c, depends upon the substructure containing the mutation
Authors: Hammack Barbara , Attneld Kathleen , Clayton Daniel , Dec Eric , Sarisky Catherine , Bowler Bruce E. , Dong Aichun 
Published: February 2009

AbstractHydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso‐1‐cytochrome c. Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine. Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy. Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the m‐value, the rate of change of free energy with respect to denaturant concentration, to different degrees. Changes in m‐values are indicative of changes in the equilibrium folding mechanism of a protein. Decreases in m‐values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state. When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the m‐value for a given variant appear to depend upon the subtructure in which the mutation is made. These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein.
Efficient sequence analysis of the six gene products (7‐74 kDA) from the escherichia coli thiamin biosynthetic operon by tandem high‐resolution mass spectrometry
Authors: Kelleher Neil L. , Taylor Sean V. , Grannis David , Kinsland Cynthia , Chiu Hsiu‐Ju , Begley Tadhg P. , Mclafferty Fred W. 
Published: February 2009

AbstractThe 105resolving power and MS/MS capabilities of Fourier‐transform mass spectrometry provide electrospray ionization mass spectra containing > 100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA‐derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply‐charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C‐terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.
A superfamily of metalloenzymes unifies phosphopentomutase and cofactor‐independent phosphoglycerate mutase with alkaline phosphatases and sulfatases
Authors: Galperin Michael Y. , Koonin Eugene V. , Bairoch Amos 
Published: February 2009

AbstractSequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal‐binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N‐acetylgalactosamine‐4‐sulfatase, and cerebroside sulfatase, enzymes with known three‐dimensional structures, with phosphopentomutase, 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin‐6‐phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC‐1, and several closely related sulfatases. In addition to the metal‐binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux‐Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.
Overexpression of recombinant proteins with a C‐terminal thiocarboxylate: Implications for protein semisynthesis and thiamin biosynthesis
Authors: Kinsland Cynthia , Taylor Sean V. , Kelleher Neil L. , Mclafferty Fred W. , Begley Tadhg P. 
Published: February 2009

AbstractA facile and rapid method for the production of protein C‐terminal thiocarboxylates on DNA‐encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein‐containing fusion proteins, can produce multi‐milligram quantities of protein C‐terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.
Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics
Authors: Rajpal Arvind , Taylor Marc G. , Kirsch Jack F. 
Published: February 2009

AbstractThe hen (chicken) egg‐white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL‐I0 Fab (Fab‐I0) was investigated by alanine scan mutagenesis. The association rate constants (kon) for the HEWL.Fab‐10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of kon fall within a factor of 2 of the wild‐type (WT) HEWL value (1.43 ± 0.13 × l06 M−1 s−1), while the increases in koff more nearly reflect the total change in free energies of the complex (ΔΔGD). The dissociation constants (KD) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL Fab‐I0 complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and Dl0lA antibody complexes are moderately destabilized (0.7 > ΔΔGD < 1.0 kcal/mol). Additional mutations of the “hotspot” residues (TYRO, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the ϵ‐amino group of Lys96, contribute to the stability of the complex. The value of ΔΔGD for the R21A mutant complex is a distinct outlier in the Arg21 replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.
Effects of salt bridges on protein structure and design
Authors: Sindelar Charles V. , Hendsch Zachary S. , Tidor Bruce 
Published: February 2009

AbstractTheoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211‐226) and experiments (Waldburger CD et al., 1995, Nut Struct Biol 2:122‐128; Wimley WC et al., 1996, Proc Natl Acud Sci USA 93:2985‐2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To examine the effect of salt bridges on protein structure and stability in more detail, we have developed an energy function for simple cubic lattice polymers based on continuum electrostatic calculations of a representative selection of salt bridges found in known protein crystal structures. There are only three types of residues in the model, with charges of ‐1, 0, or +1. We have exhaustively enumerated conformational space and significant regions of sequence space for three‐dimensional cubic lattice polymers of length 16. The results demonstrate that, while the more highly charged sequences are less stable, the loss of stability is accompanied by a substantial reduction in the degeneracy of the lowest‐energy state. Moreover, the reduction in degeneracy is greater due to charges that pair than for lone charges that remain relatively exposed to solvent. We have also explored and illustrated the use of ion‐pairing strategies for rational structural design using model lattice studies.
Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr
Authors: Kim Seho , Baum Jean 
Published: February 2009

Abstractα‐Lactalbumin (α‐LA) undergoes a pH‐dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pigα‐LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation inα‐LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation ofα‐LA are concentrated in the calcium binding region of the protein.
Structural/functional properties of the Glu1‐HSer57 N‐terminal fragment of human plasminogen: Conformational characterization and interaction with kringle domains
Authors: An Seong Soo A. , Marti Daniel N. , Llinas Miguel , Carreño Cristina , Albericio Fernando , Schaller Johann 
Published: February 2009

AbstractThe Glu1‐Val79 N‐terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of ˜9 kDa each. K1, K2, K4, and K5 contain each a lysine‐binding site (LBS). Pgn was cleaved with CNBr and the Glu1‐HSer57 N‐terminal fragment (CB‐NTP) isolated. In addition, the Ile27‐Ile56 peptide (L‐NTP) that spans the doubly S‐S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB‐NTP with each of the Pgn kringles were monitored by 1H‐NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rKl, Ka ˜ 4.6 mM−1; rK2, Ka ˜ 3.3 mM−1; K4, Ka ˜ 6.2 mM−1; K5, KMa ˜ 2.3 mM−1. Thus, the lysine‐binding kringles interact with CB‐NTP more strongly than with Nα‐acetyl‐L‐lysine methyl ester (Ka < 0.6 mM−1), which reveals specificity for the NTP. In contrast, CB‐NTP does not measurably interact with rK3, which is devoid of a LBS.CB‐NTP and L‐NTP 1H‐NMR spectra were assigned and interproton distances estimated from 1H‐1H Overhauser (NOESY) experiments. Structures of L‐NTP and the Glu1‐Ile27 segment of CB‐NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB‐NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6‐9, 12‐16, 28‐30, and 45‐48. Within the Cys34‐Cys42 loop of L‐NTP, the structure of the Glu‐Glu‐Asp‐Glu‐Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys50 within the Cys42‐Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glu1‐Pgn. Overall, the CB‐NTP and L‐NTP fragments are of low regular secondary structure content — as indicated by UV‐CD spectra — and exhibit fast amide 1H‐2H exchange in 2H2O, suggestive of high flexibility.
Human nucleotide excision repair protein XPA: Extended X‐ray absorption fine‐structure evidence for a metal‐binding domain
Authors: Hess Nancy J. , Buchko Garry W. , Ni Shuisong , Thrall Brian D. , Kennedy Michael A. , Conradson Steven D. , Espinosa F. Javier 
Published: February 2009

AbstractThe ubiquitous, multi‐enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc‐associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4‐type motif, C10S‐X2‐C108‐X17‐C126‐X2‐C129, located in the minimal DNA binding region of XPA (M98‐F219). The zinc‐associated motif is essential for damaged DNA recognition. Extended X‐ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA‐binding domain of XPA (ZnXPA‐MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn‐S bond length of 2.34 ± 0.01 Å. XPA‐MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue‐colored protein that was primarily (>95%) cobalt associated (CoXPA‐MBD). EXAFS spectra collected on CoXPA‐MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co‐S bond length of 2.33 ± 0.02 Å.
Architecture of β‐barrel membrane proteins: Analysis of trimeric porins
Authors: Seshadri K. , Garemyr Robert , Wallin Erik , Heijne Gunnar Von , Elofsson Arne 
Published: February 2009

AbstractWe have analyzed the known three‐dimensional structures of trimeric porins from bacterial outer membranes. The distribution of surface‐exposed residues in a direction perpendicular to the membrane is similar to that in helical membrane proteins, with aliphatic residues concentrated in the central 20 Å of the bilayer. Outside these residues is a layer of aromatic residues, followed by polar and charged residues. Residues in the trimer interface are more conserved than residues not in the interface. By comparing the interface and noninterface residues, an interface preference scale has been derived that may be used as a basis for predicting interface surfaces in monomer models.