bluuebunny/crossref_metadata_2025 huggingface dataset:
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

We 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

The 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

The 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

Three 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

Hydrophilic 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

The 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

Sequence 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

A 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

The 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

Theoretical 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

α‐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

The 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

The 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

We 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.
Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity
Authors: Sanschagrin Paul C. , Kuhn Leslie A.
Published: February 2009

Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.
Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy
Authors: Couprie Joel , Remerowski M. Lyndsay , Bailleul Arnaud , Courçon Marie , Gilles Nicolas , Quéméneur Eric , Jamin Nadege
Published: February 2009

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13Cα, 13CO, 1Hα, and 13Cß NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety‐nine percent of the frequencies were assigned using a combination of triple (1H‐13C‐15N) and double resonance (1H‐15N or 1H‐13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3JHNHα and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13Cα chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.
Structure of the xylanase from Penicillium simplicissimum
Authors: Schmidt Andrea , Kratky Christoph , Schlacher Anton , Schwab Helmut , Steiner Walter
Published: February 2009

Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X‐ray data were collected at cryo‐temperature to a Crystallographic resolution of 1.75 Å. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (Rfree = 23%) for data between 10 and 1.75 Å. The xylanase folds in an (α/β)8 barrel (TIM‐barrel), with additional helices and loops arranged at the “top” forming the active site cleft. In its overall shape, the P simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.
Tertiary structure‐dependence of misfolding substitutions in loops of the maltose‐binding protein
Authors: Raffy Sébastien , Sassoon Nathalie , Hofnung Maurice , Betton Jean‐Michel
Published: February 2009

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the αI/βB loop, of the N‐domain that are critical for in vivo folding of the maltose‐binding protein (MalE31). The tertiary context‐dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent αβ loop of the C‐domain to the same amino acid substitutions (MaIE219). Moving the loop mutation from the N‐ to the C‐domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates inEscherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off‐pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super‐secondary structures of the N‐domain is a rate‐limiting step in the folding pathway of MalE.
Calcium‐binding and structural stability of echidna and canine milk lysozymes
Authors: Kikuchi Masayuki , Kawano Keiichi , Nitta Katsutoshi
Published: February 2009

For echidna and canine milk lysozymes, which were presumed to be the calcium‐binding lysozymes by their amino acid sequences, we have quantitated their calcium‐binding strength and examined their guanidine unfolding profiles.

The calcium‐binding constants of echidna and canine lysozymes were determined to be 8.6 × 106 M−1 and 8.9 X 106 M−1 in 0.1 M KCl at pH 7.1 and 20 °C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of α‐lactalbumin, through a stable molten globule intermediate. However, neither calcium‐bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg‐white lysozyme, avian calcium‐binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel‐Boston‐Berlin: Birkhauzer Verlag. pp 9‐31) that the lineage of avian and echidna calcium‐binding lysozymes and that of eutherian calcium‐binding lysozymes diverged separately from that of conventional lysozymes.
The functional importance of structural differences between the mannitol‐specific IIAmannitol and the regulatory IIAnitrogen
Authors: Van Montfort Rob L. M. , Dijkstra Bauke W.
Published: February 2009

The three‐dimensional structures of the IIA domain of the mannitol‐specific phosphoenol‐pyruvate dependent phos‐photransferase system (PTS) of Escherichia coli and the regulatory IIAntr enzyme have been compared. The enzymes are 20% identical in sequence and contain the sequence motif specific for IIA proteins belonging to the mannitol‐fructose family of the PTS. The fold of the enzymes is nearly identical, which confirms their evolution from a common ancestor. Moreover, the phosphorylation site of IIAmtl (His65) and one of the observed conformations of the active site Arg49 are extremely similar to their equivalents (His73 and Arg57) in IIAntr. In contrast, His l20, the equivalent of the second active site His111 of IIAmtl, is not located in the active site of IIAntr but points into the solvent on the other side of the molecule. The different position of His120 makes it unlikely that this residue assists in phosphoryl transfer in the regulatory IIAntrs. As His120 is conserved in all IIAntr enzymes, it could have an essential role in the recognition of the target protein of IIAntr.
Mapping the lifetimes of local opening events in a native state protein
Authors: Kragelund Birthe B. , Heinemann Bo , Knudsen Jens , Poulsen Flemming M.
Published: February 2009

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four‐helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s.In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h.For these the lifetime in the open state is also around 1 s.The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding.The results suggest a mechanism of hydrogen exchange by progressive local openings.
Semisynthesis of cytotoxic proteins using a modified protein splicing element
Authors: Evans Thomas C. , Benner Jack , Xu Ming‐Qun
Published: February 2009

Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C‐terminus of a recombinant protein. Nucleophilic attack on this thioester by the N‐terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol‐induced cleavage of the precursor led to the liberation of the target protein with a C‐terminal thioester‐tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full‐length products that displayed catalytic activity indicative of the wild‐type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8. 2 s‐1 and 1. 5 mM, in good agreement with reported values of 8. 3 s‐1 and 1. 2 mM (Hodges&Merrifield, 1975). Ligated HpaI had a specific activity of 0. 5–1. 5 × 106 U/mg, which compared favorably with the expected value of 1–2 × 106 U/mg (J. Benner, unpubl. obs. ). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.
Solution structures of stromelysin complexed to thiadiazole inhibitors
Authors: Stockman Brian J. , Waldon Daniel J. , Gates Jo A. , Scahill Terrence A. , Kloosterman David A. , Mizsak Stephen A. , Jacobsen E. Jon , Belonga Kenneth L. , Mitchell Mark A. , Mao Boryeu , Petke James D. , Goodman Linda , Powers Elaine A. , Ledbetter Steven R. , Kaytes Paul S. , Vogeli Gabriel , Marshall Vincent P. , Petzold Gary L. , Poorman Roger A.
Published: February 2009

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase, have been implicated in several pathological conditions including arthritis and cancer. Small‐molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/inhibitor complexes have been investigated using H, 13C, and 15N NMR spectroscopy. Both inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1‐S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring‐flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring‐flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.
Singular points of protein β‐sheets
Authors: Liu Wei‐Min , Chou Kuo‐Chen
Published: February 2009

Protein ß‐sheets can be regarded as surfaces. Two surfaces can be connected along a common edge to form a larger surface, or two edges of a surface can coalesce to form a closed sheet such as a ß‐barrel. Singular points are locations where these connections are not perfect. In protein ß‐sheets, a singular point is characterized by a residue separating two ß‐ladders. In this paper, we study the singular points of protein ß‐sheets from the surface topologic viewpoint, summarize our search results from the protein structural data in the Protein Data Bank, and present examples where singular points are near the active sites and may contribute to forming the proper relative positions of catalytic residues.
Role of the electrostatic loop charged residues in Cu, Zn superoxide dismutase
Authors: Polticelli Fabio , Battistoni Andrea , O'Neill Peter , Rotilio Giuseppe , Desideri Alessandro
Published: February 2009

We have expressed and characterized a mutant of Xenopus laevis Cu, Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 → Leu, Asp130 → Gln, Glu131 → Gln, and Lys134 → Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6. 7 × 109 M−1 s−1, at pH 7 and μ = 0. 02 M). Brownian dynamics simulations give rise to identical enzyme‐substrate association rates for both wild‐type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild‐type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.
Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry
Authors: Zaia Joseph , Fabris Daniele , Wei Dong , Karpel Richard L. , Fenselau Catherine
Published: February 2009

The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil‐alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc‐bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single‐stranded DNA.
The orientation and dynamics of substance P in lipid environments
Authors: Keire David A. , Kobayashi Mitsuo
Published: February 2009

The membrane‐associated conformation of substance P (RPKPQQFFGLM‐NH2) has been previously proposed to be the NK1‐receptor‐active conformation. In this work, NMR methods are applied to explore the orientation and dynamics of substance P at lipid surfaces for which the peptide's three‐dimensional structure had been previously determined. Here the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS) micelles has been found to cause sequence specific changes in the acid‐ and base‐catalyzed amide proton exchange rates relative to the solution state values. On binding of substance P to SDS micelles, the FFG portion showed the largest decreases in the base‐catalyzed amide exchange rates. Similar sequence‐specific changes in substance P are observed in the presence of DPC micelles, albeit at much weaker levels due to fast exchange between free and bound forms of the peptide. These differences are attributed to the location of the amide protons either in the surface double layer (via electrostatic effect) or inserted into the polar head group region of the micelles (via low dielectric). The sequence‐specific effects of micelle association were also observed in the homonuclear nonselective spin‐lattice relaxation time; these, in combination with spin‐spin relaxation times, were used to calculate correlation times for the backbone amide protons. These data combined with paramagnetic broadening observations on peptide protons in the presence of spin‐labeled lipids yield a detailed model of the interaction of substance P with lipid surfaces.
Cation‐π (Na+‐Trp) interactions in the crystal structure of tetragonal lysozyme
Authors: Wouters Johan
Published: February 2009

Experimental evidence of a cation‐π interaction between a sodium cation (Na+) and the indole ring of residue Trpl23 in a structure (2. 0 Å) of hen egg‐white lysozyme is presented. The geometry of the metal ion‐π interaction observed in the protein structure (distance between the aromatic plane and the cation ∼4 Å) is consistent with geometries observed among small molecules crystal structures and quantum chemistry ab initio calculations. The present crystal structure of lysozyme provides unique structural information about the geometry of binding of cations to π systems in proteins. It shows that the metal ion‐π interaction within proteins is not significantly different from similar bindings found in small molecules and that it can be modeled by theoretical methods.
Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins
Authors: Lerche Mathilde H. , Poulsen Flemming M.
Published: February 2009

The structure of a nonspecific lipid transfer protein from barley (ns‐LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns‐LTPbarley in the absence of palmitate, to the structure of ns‐LTPbarley in complex with palmitoyl coenzyme A, to the structure ofns‐LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns‐LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.
Crystal structure of the effector‐binding domain of the trehalose‐repressor of escherichia coli, a member of the LacI family, in its complexes with inducer trehalose‐6‐phosphate and noninducer trehalose
Authors: Hars Ulrike , Horlacher Reinhold , Boos Winfried , Welte Wolfram , Diederichs Kay
Published: February 2009

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose‐6‐phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 Å resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 Å resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central β‐sheet sandwiched between α‐helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose‐6‐phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose‐6‐phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.
The crystal structure of NADPH: Ferredoxin reductase from azotobacter vinelandii
Authors: Prasad G. Sridhar , Kresge N. , Muhlberg A. B. , Shaw A. , Stout C. D. , Jung Y. S. , Burgess B. K.
Published: February 2009

NADPH: ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 Å resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N‐terminal six‐stranded antiparallel β‐barrel domain, which binds FAD, and a C‐terminal five‐stranded parallel β‐sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C‐terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C‐terminus by three residues relative to EcFldR and by four residues relative to FNR. The C‐terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.
Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme‐MgADP‐AlF3‐diaminopelargonic acid and enzyme‐MgADP‐dethiobiotin‐phosphate; implications for catalysis
Authors: Käck Helena , Sandmark Jenny , Schneider Gunter , Lindqvist Ylva , Gibson Katharine J.
Published: February 2009

The crystal structures of two complexes of dethiobiotin synthetase, enzyme‐diaminopelargonic acid‐MgADP‐AlF3 and enzyme‐dethiobiotin‐MgADP‐Pi, respectively, have been determined to 1.8 Å resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two‐metal mechanism for this enzyme.