bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractTo 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractCytochrome 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

AbstractComplete 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

AbstractCore‐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

AbstractThe 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

AbstractThe 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

AbstractThe 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.