bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between protein structure and geometrical constraints
Authors: Lund Ole , Hansen Jan , Brunak Søren , Bohr Jakob
Published: February 2009

We evaluate to what extent the structure of proteins can be deduced from incomplete knowledge of disulfide bridges, surface assignments, secondary structure assignments, and additional distance constraints. A cost function taking such constraints into account was used to obtain protein structures using a simple minimization algorithm. For small proteins, the approximate structure could be obtained using one additional distance constraint for each amino acid in the protein. We also studied the effect of using predicted secondary structure and surface assignments. The constraints used in this approach typically may be obtained from low‐resolution experimental data. When using a cost function based on distances, half of the resulting structures will be mirrored, because the resulting structure and its mirror image will have the same cost. The secondary structure assignments were therefore divided into chirality constraints and distance constraints. Here we report that the problem of mirrored structures, in some cases, can be solved by using a chirality term in the cost function.
Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N‐ and C‐termini regions of the ROMK1 K+ channel
Authors: Ben‐Efraim Iris , Shai Yechiel
Published: February 2009

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post‐M2). Peptides that correspond to M0, post‐M2, and a control peptide, pre‐M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self‐assemble and coassemble in the membrane‐bound state. CD spectroscopy revealed that both M0 and post‐M2 adopt highly α‐helical structures in 1% SDS and 40% TFE/water, whereas pre‐M0 is not α‐helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD‐labeled peptides demonstrated that both M0 and post‐M2, but not pre‐M0, bind to zwitterionic phospholipid membranes with partition coefficients of 103–105 M−1. A surface localization for both post‐M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that M0 and post‐M2 can coassemble in their membrane‐bound state, but cannot self‐associate when membrane‐bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994, Science 264:844–847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039–1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane‐bound polypeptides are also discussed.
Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification
Authors: Bray Mark R. , Gilkes Neil R. , Kilburn Douglas G. , Warren R. Antony J. , Mcintosh Lawrence P. , Johnson Philip E.
Published: February 2009

The cellulose‐binding domain (CBDCex) of the mixed function glucanase‐xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the β‐barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993–7009). Although all five tryptophans can be oxidized by N‐bromosuccinimide (NBS), stopped‐flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604–607], each of the exposed tryptophans interacts reversibly with cellulose.
The C2 domain calcium‐binding motif: Structural and functional diversity
Authors: Nalefski Eric A. , Falke Joseph J.
Published: February 2009

The C2 domain is a Ca2+‐binding motif of approximately 130 residues in length originally identified in the Ca2+‐dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid‐second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin I and phosphoinositide‐specific phospholipase C‐δ1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.
Protein structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme
Authors: Vetter Ingrid R. , Baase Walter A. , Heinz Dirk W. , Xiong Jian‐Ping , Snow Sheila , Matthews Brian W.
Published: February 2009

To further investigate the ways in which proteins respond to changes in the length of the polypeptide chain, a series of 32 insertions and five deletions were made within nine different α‐helices of T4 lysozyme. In most cases, the inserted amino acid was a single alanine, although in some instances up to four residues, not necessarily alanine, were used. Different insertions destabilized the protein by different amounts, ranging from approximately 1 to 6 kcal/mol. In one case, no protein could be obtained. An “extension” mutant in which the carboxy terminus of the molecule was extended by four alanines increased stability by 0.3 kcal/mol. For the deletions, the loss in stability ranged from approximately 3 to 5 kcal/mol. The structures of six insertion mutants, as well as one deletion mutant and the extension mutant, were determined, three in crystal forms nonisomorphous with wild type. In all cases, including previously described insertion mutants within a single α‐helix, there appears to be a strong tendency to preserve the helix by translocating residues so that the effects of the insertion are propagated into a bend or loop at one end or the other of the helix. In three mutants, even the hydrophobic core was disrupted so as to permit the preservation of the α‐helix containing the insertion. Translocation (or “register shift”) was also observed for the deletion mutant, in this case a loop at the end of the helix being shortened. In general, when translocation occurs, the reduction in stability is only moderate, averaging 2.5 kcal/mol. Only in the most extreme cases does “bulging” or “looping‐out” occur within the body of an α‐helix, in which case the destabilization is substantial, averaging 4.9 kcal/mol. Looping‐out can occur for insertions close to the end of a helix, in which case the destabilization is less severe, averaging 2.6 kcal/mol. Mutant A73‐[AAA], as well as mutants R119‐[A] and V131‐[A], include shifts in the backbone of 3–6 Å, extending over 20 residues or more. As a result, residues 114–142, which form a “cap” on the carboxy‐terminal domain, undergo substantial reorganizations such that the interface between this “cap” and the rest of the protein is altered substantially. In the case of mutant A73‐[AAA], two nearby α‐helices, which form a bend of approximately 105 degrees in the wild‐type structure, reorganize in the mutant structure to form a single, essentially straight helix. These structural responses to mutation demonstrate the plasticity of protein structures and illustrate ways in which their three‐dimensional structures might change during evolution.
Strategy to design peptide inhibitors: Structure of a complex of proteinase K with a designed octapeptide inhibitor N‐Ac‐Pro‐Ala‐Pro‐Phe‐DAla‐Ala‐Ala‐Ala‐NH2 at 2.5 Å resolution
Authors: Saxena Ajay K. , Singh Tej P. , Peters Klaus , Fittkau Siegfried , Betzel Christian
Published: February 2009

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N‐Ac‐Pro‐Ala‐Pro‐Phe‐DAla‐Ala‐Ala‐Ala‐NH2, has been determined at 2.5 Å resolution and refined to an R‐factor of 16.7% for 7,430 reflections in the resolution range of 8.0–2.50 Å. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydrolyzed between Phe4I and DAla5I (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 Å between the carbonyl carbon of Phe4I and the main‐chain nitrogen of DAla5I. The N‐terminal tetrapeptide occupies subsites S1‐S5 (S5 for acetyl group), whereas the C‐terminal part fits into S1′‐S5′ region (S5′ for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octapeptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.
A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I
Authors: Matsuo Yo , Yamada Atsuko , Tsukamoto Kikuo , Tamura Hiro‐Omi , Ikezawa Hiroh , Nakamura Haruki , Nishikawa Ken
Published: February 2009

The three‐dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site‐directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase‐sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.
A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content
Authors: Borén Krishna , Freskgård Per‐Ola , Carlsson Uno
Published: February 2009

The CD spectra of human carbonic anhydrase I and II and bovine carbonic anhydrase III were recorded and analyzed. The 3D structures of these isoenzymes are known, showing very similar secondary structure and polypeptide‐chain fold. The tryptophan content, however, differs between the isoenzymes, i.e., isoenzymes I, II, and III possess 6, 7, and 8 tryptophans, respectively. All of the tryptophans except the additional tryptophans in isoenzymes II and III, i.e., W245 and W47, are conserved. Despite the fact that X‐ray structure determinations showed that the isoenzymes had highly similar secondary structure, the contents of α‐helix and β‐sheet structure differed considerably when using different CD algorithms for estimation of the fractions of various secondary structural elements. This shows that aromatic amino acids also interfere in the wavelength region (far‐UV) used to calculate the amount of secondary structure. Such interference is especially problematic when analyzing proteins like carbonic anhydrase, which consist mainly of β‐structure that gives rise to weak ellipticity bands, compared to the bands arising from α‐helical structure.
NMR assignments and relaxation studies of Thiobacillus versutus ferrocytochrome c‐550 indicate the presence of a highly mobile 13‐residues long C‐terminal tail
Authors: Ubbink Marcellus , Pfuhl Mark , Oost John Van Der , Berg Axel , Canters Gerard W.
Published: February 2009

Cytochrome c‐550 of Thiobacillus versutus functions as an electron transfer protein in a chain of redox proteins that enables T. versutus to grow on methylamine. It is a single‐heme protein of 134 residues, related to mitochondrial cytochrome c. Cytochrome c‐550, as well as several other bacterial c2‐type cytochromes, contain a C‐terminal extension of 13–16 amino acids of unknown function, compared to mitochondrial cytochrome c.

NMR experiments were performed to obtain structural and dynamic information on the protein in solution. For this purpose, T. versutus cytochrome c‐550 was labeled with 15N and 13C using 13C‐methanol grown Paracoccus denitrificans as a host for heterologous expression. NMR assignments were obtained for the 1H, 15N, and 13C nuclei in the backbone and the β‐positions of the protein and the secondary structure was determined. 15N‐relaxation studies were performed to characterize the dynamic properties of the protein. The results indicate that the main part of T. versutus ferrocytochrome c‐550 exists in solution as a rigid, well‐ordered molecule with a secondary structure that is very similar to that of P. denitrificans cytochrome c‐550, as observed in crystals. The C‐terminal extension, however, is unstructured and highly mobile. The possible origin and function of the extension are discussed.
Direct evidence for the localization of the steroid‐binding site of the plasma sex steroid‐binding protein (SBP or SHBG) at the interface between the subunits
Authors: Sui Li‐Ming , Hughes William , Hoppe Agnes J. , Pétra Philip H.
Published: February 2009

Complete dissociation of dimeric plasma sex steroid‐binding protein (SBP or SHBG) was obtained in 6 M urea at 10 °C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca++ or steroid. Renatured monomers yield dimers with dissociation constants for 5α‐dihydrotesterone (DHT) and 17β‐estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid‐binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often‐asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid‐binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
NMR studies of structure, hydrogen exchange, and main‐chain dynamics in a disrupted‐core mutant of thioredoxin
Authors: Lorimier Robert De , Hellinga Homme W. , Spicer Leonard D.
Published: February 2009

Core‐packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core‐packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two‐ and three‐dimensional NMR. Chemical shift differences between the mutant and wild‐type main‐chain resonances reveal that structural changes caused by the mutation are localized within 12 Å of the altered side chain. The majority of resonances are unchanged, as are many 1H‐1H NOEs indicative of the main‐chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central β‐sheet of both mutant and wild‐type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main‐chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main‐chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout.
Helix propagation and N‐cap propensities of the amino acids measured in alanine‐based peptides in 40 volume percent trifluoroethanol
Authors: Rohl Carol A. , Chakrabartty Avijit , Baldwin Robert L.
Published: February 2009

The helix propagation and N‐cap propensities of the amino acids have been measured in alanine‐based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix‐stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine‐based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N‐capping explicitly included in the helix‐coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all β‐branched and β‐substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, β‐substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (ΔΔG) between alanine and other nonpolar amino acids are twice as large in water as predicted from side‐chain conformational entropies, but ΔΔG values in 40% TFE are close to those predicted from side‐chain entropies. This dependence of ΔΔG on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N‐cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N‐cap values shown by the amino acids.
Saccharomyces cerevisiae mitochondria lack a bacterial‐type Sec machinery
Authors: Glick Benjamin S. , Heijne Gunnar Von
Published: February 2009

The bacterial Sec genes encode a generalized protein export machinery. Although the mitochondria present in eukaryotic cells are derived from bacterial ancestors, a comprehensive search of the complete genomic sequence for the eukaryotic yeast Saccharomyces cerevisiae did not reveal any close homologs of the bacterial Sec genes, strongly suggesting that yeast mitochondria lack a generalized bacterial‐type export system. This finding has implications for the sorting of imported mitochondrial proteins to the intermembrane space compartment, and also for the insertion of mitochondrially encoded proteins into the inner membrane.
Characterization of the allosteric binding pocket of human liver fructose‐1,6‐bisphosphatase by protein crystallography and inhibitor activity studies
Authors: Iversen L. F. , Brzozowski M. , Hastrup S. , Hubbard R. , Kastrup J. S. , Larsen I. K. , Nærum L. , Nørskov‐Lauridsen L. , Rasmussen P. B. , Thim L. , Wiberg F. C. , Lundgren K.
Published: February 2009

The structures of three complexes of human fructose‐1,6‐bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5‐Amino‐4‐carboxamido‐1β‐D‐5‐phosphate‐ribofuranosyl‐1H‐imidazole (AICAR‐P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR‐P is rotated 180° compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR‐P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR‐P were determined to be 1.7, 1.4, and 20.9 μ, respectively. Thus, the approximately 10 times lower potency of AICAR‐P is difficult to explain solely from the variations observed in the binding pocket.

Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR‐P complexes. This implies an important role of the conserved water molecule in binding of the ligand.
Predicted structure of the extracellular region of ligand‐gated ion‐channel receptors shows SH2‐like and SH3‐like domains forming the ligand‐binding site
Authors: Gready Jill E. , Ranganathan Shoba , Schofield Peter R. , Matsuo Yo , Nishikawa Ken
Published: February 2009

Fast synaptic neurotransmission is mediated by ligand‐gated ion‐channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand‐binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3receptors) or anions (GABAAand glycine receptors and the invertebrate glutamate‐binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D‐3D structure mapping approach, we have modeled the extracellular ligand‐binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium‐resolution electron microscopy structures and with experimental structure/function data from ligand‐binding, antibody‐binding, mutagenesis, protein‐labeling and subunitlinking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor α1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand‐binding domain with the activation of the M2 transmembrane helix is also suggested.
Template‐based docking of a prolactin receptor proline‐rich motif octapeptide to FKBP12: Implications for cytokine receptor signaling
Authors: Soman K. V. , Hanks B. A. , Tien H. , Chari M. V. , O'Neal K. D. , Morrisett J. D.
Published: February 2009

A conserved proline‐rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu‐Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282–284). We have docked the prolactin receptor PRM (Ile1‐Phe2‐Pro3‐Pro4‐Val5‐Pro6‐Gly7‐Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833–844), energy minimization of the octapeptide conformers so obtained, template‐based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506‐peptide hybrid bound to FKBP12. Val5‐Pro6 of the PRM was taken to be the biologically relevant Xaa‐Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7 → O4 and N2H2 → O8, and two intermolecular ones, Ile56: N‐H → O=C:Pro6 and Tyr82:O‐H → O=C:Gly7. This conformer features a Type I β‐turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis‐trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.
Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli
Authors: Bravo Jeronimo , Fita Ignacio , Ferrer Juan C. , Ens Werner , Hillar Alex , Switala Jacek , Loewen Peter C.
Published: February 2009

A bond between the Nδ of the imidazole ring of His 392 and the Cβ of the essential Tyr 415 has been found in the refined crystal structure at 1.9 Å resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix‐assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the Cβ atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392‐Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80° relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self‐catalyzed heme conversion reaction and the formation of the His‐Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound I is proposed.
Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli
Authors: Vickery Larry E. , Silberg Jonathan J. , Ta Dennis T.
Published: February 2009

The hscA and hscB genes of Escherichia coli encode novel chaperone and co‐chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentrations ˜20 μM and ˜10 μM, respectively. The levels do not change appreciably following heat shock (44°C), but a small increase in Hsc20 is observed following a shift to 10°C. Purified Hsc66 exhibits a low intrinsic ATPase activity (˜0.6 min−1 at 37° C), and Hsc20 was found to stimulate this activity up to 3.8‐fold with half‐maximal stimulation at a concentration ˜5 μM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non‐stress conditions.
Consistency in structural energetics of protein folding and peptide recognition
Authors: Zhang Chao , Cornette James L. , Delisi Charles
Published: February 2009

We report a new free energy decomposition that includes structure‐derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single‐domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single‐domain proteins for which the three‐dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (ΔSbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence‐dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA‐A2.1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.
Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme
Authors: Bhattacharjya Surajit , Balaram Padmanabhan
Published: February 2009

A molten globule‐like state of hen egg‐white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native‐like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native‐like tryptophan fluorescence quenching. 1‐D NMR spectra indicates loss of native‐like tertiary fold as evident from the absence of ring current‐shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule‐like state to an “open” helical state is observed at higher concentrations of HFA (≥50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side‐chain‐side‐chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four α‐helices along with the C‐terminal 310 helix, whereas little or no protection is observed for most of the amide protons from the triple‐stranded antiparallel β‐sheet domain. This equilibrium molten globule‐like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for α‐lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.
Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase
Authors: Peretz Moshe , Weiner Lev M. , Burstein Yigal
Published: February 2009

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1‐oxy‐2,2,5,5‐tetramethyl‐3‐imidazoline‐4‐yl)disulfide, via a thiol‐disulfide exchange reaction and with 2[14C]iodoacetic acid, via S‐alkylation. The respective reactions were monitored by electron paramagnetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S‐carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o‐phenanthroline. This cysteine residue, which could serve as a putative ligand to the active‐site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of ≤ 10 Å between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 °C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.
Hydrogen exchange: The modern legacy of Linderstrøm‐Lang
Authors: Englander S. W. , Mayne L. , Bai Y. , Sosnick T. R.
Published: February 2009

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm‐Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.
The uncharged surface features surrounding the active site ofEscherichia coliDsbA are conserved and are implicated in peptide binding
Authors: Guddat Luke W. , Martin Jennifer L. , Bardwell James C.A. , Zander Thomas
Published: February 2009

DsbA is a protein‐folding catalyst from the periplasm ofEscherichia colithat interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Å, and present a proposal for its interaction with peptide.

The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified—the type IV β‐turn Phe 63‐Met 64‐Gly 65‐Gly 66, which connects the two domains.

Three unique features on the active site surface of the DsbA molecule—a groove, hydrophobic pocket, and hydrophobic patch—form an extensive uncharged surface surrounding the active‐site disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active‐site disulfide are uncharged in all nine DsbA proteins.

A model for DsbA‐peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active‐site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.
MultiCoil: A program for predicting two‐and three‐stranded coiled coils
Authors: Wolf Ethan , Kim Peter S. , Berger Bonnie
Published: February 2009

A new multidimensional scoring approach for identifying and distinguishing trimeric and dimeric coiled coils is implemented in the MultiCoil program. The program extends the two‐stranded coiled‐coil prediction program PairCoil to the identification of three‐stranded coiled coils. The computations are based upon data gathered from a three‐stranded coiled‐coil database comprising 6,319 amino acid residues, as well as from the previously constructed two‐stranded coiled‐coil database. In addition to identifying coiled coils not predicted by the two‐stranded database programs, MultiCoil accurately classifies the oligomerization states of known dimeric and trimeric coiled coils. Analysis of the MultiCoil scores provides insight into structural features of coiled coils, and yields estimates that 0.9% of all protein residues form three‐stranded coiled coils and that 1.5% form two‐stranded coiled coils. The MultiCoil program is available at http://theory.Ics.mit.edu/multicoil.
Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins
Authors: Ladenstein Rudolf , Karshikoff Andrej D. , Spassov Velin Z.
Published: February 2009

The three‐dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte‐Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side‐chain conformations. Similar to the optimization of the charge‐charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 41516‐1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.
Refined structure of villin 14T and a detailed comparison with other actin‐severing domains
Authors: Wagner Gerhard , Markus Michelle A. , Matsudaira Paul
Published: February 2009

Villin 14T is the amino terminal actin monomer binding domain from the actin‐severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin‐severing proteins. Protein Science 3:70‐81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin‐severing domain, severin domain 2.
Characterization of the receptor binding determinants of granulocyte colony stimulating factor
Authors: Young Dennis C. , Cheng Qi‐Lin , Hou Jinzhao , Matthews David J. , Zhan Hangjun
Published: February 2009

We performed a series of experiments using alanine‐scanning mutagenesis to locate side chains within human granulocyte colony‐stimulating factor (G‐CSF) that are involved in human G‐CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G‐CSF. The G‐CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G‐CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild‐type G‐CSF and the E19A mutant were expressed in E. coli. The re‐folded proteins were found to have proliferative activities similar to the analagous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G‐CSF. Although G‐CSF is likely to signal through receptor homo‐dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self‐antagonism at high G‐CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G‐CSF probably does not signal via a pure 2.1 receptor:ligand complex. Thus, G‐CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high‐affinity interaction with its receptor.
Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild‐type enzyme, the I11OR mutant, and the C‐terminal DNA binding domain in solution
Authors: Pan Borlan , Ghosh Susmita , Mullen Gregory P. , Deng Zhengwu , Liu Dingjiang
Published: February 2009

γδ Resolvase is a site‐specific DNA recombinase (Mr 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low‐salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten ertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA‐induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I11OR resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I11OR mutant contains a folded catalytic core of ∼60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I11OR resolvase. No increase in the helicity of the linker region of I11OR resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.