bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monomer‐trimer equilibrium of the ectodomain of SIV gp41: Insight into the mechanism of peptide inhibition of HIV infection
Authors: Caffrey Michael , Kaufman Joshua , Stahl Stephen , Wingfield Paul , Gronenborn Angela M. , Clore G. Marius 
Published: February 2009

AbstractThe monomer‐trimer equilibrium of the ectodomain of SIV gp41 (residues 27–149, e‐gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e‐gp41 trimer is 3.1 × 1011 M−2. The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C‐labeled e‐gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e‐gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer‐trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.
The acid‐induced folded state of Sac7d is the native state
Authors: Bedell Jennifer L. , Mccrary Bradford S. , Edmondson Stephen P. , Shriver John W. 
Published: February 2009

AbstractSac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203–224). Both near‐ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid‐ and salt‐induced folded states have a native fold and are not molten globular. 1H, 15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid‐, and salt‐induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through‐bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid‐ and salt‐induced folded states are identical to those observed in the native. The radius of gyration of the acid‐induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X‐ray scattering. We conclude that acid‐induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid‐induced folding except that the A‐state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.
A 16‐amino acid peptide from human α2‐macroglobulin binds transforming growth factor‐β and platelet‐derived growth factor‐BB
Authors: Webb Donna J. , Roadcap David W. , Dhakephalkar Anita , Gonias Steven L. 
Published: February 2009

Abstractα2‐Macroglobulin (α2M) is a major carrier of transforming growth factor‐β (TGF‐β) in vitro and in vivo. By screening glutathione S‐transferase (GST) fusion proteins with overlapping sequences, we localized the TGF‐β‐binding site to aa 700–738 of the mature human α2M subunit. In separate experiments, we screened overlapping synthetic peptides corresponding to aa 696–777 of α2M and identified a single 16‐mer (718–733) that binds TGF‐β1. Platelet‐derived growth factor‐BB (PDGF‐BB) bound to the same peptide, even though TGF‐β and PDGF‐BB share almost no sequence identity. The sequence of the growth factor‐binding peptide, WDLVVVNSAGVAEVGV, included a high proportion of hydrophobic amino acids. The analogous peptide from murinoglobulin, a human α2M homologue that does not bind growth factors, contained only three nonconservative amino acid substitutions; however, the MUG peptide failed to bind TGF‐β1 and PDGF‐BB. These results demonstrate that a distinct and highly‐restricted site in α2M, positioned near the C‐terminal flank of the bait region, mediates growth factor binding. At least part of the growth factor‐binding site is encoded by exon 18 of the α2M gene, which is notable for a 5′ splice site polymorphism that has been implicated in Alzheimer's Disease.
For the record: Chemical modification of a variant of human MIP‐1α; implications for dimer structure
Authors: Ashfield J. T. , Fisher J. , Meyers T. , Lowne D. , Czaplewski L. G. , Dudgeon T. , Varley P. G. , Arnold J. R. P. , Tan P. , Yang J.‐C. 
Published: February 2009

AbstractA sequence variant of human MIP‐1α, in which Asp26 has been replaced by Ala, has been chemically modified by the addition of 13C‐labeled methyl groups at each of the lysine residues and the N‐terminus. The sites of methylation have been verified by a combination of MALDI‐TOF mass spectrometric experiments and tryptic digestion followed by N‐terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra‐centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP‐1α D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP‐3, rather than the elongated or cylindrical structure determined for dimers of huMIP‐1β and RANTES.
Structural characterization of a mutant peptide derived from ubiquitin: Implications for protein folding
Authors: Zerella Rosa , Williams Dudley H. , Chen Pei‐Yeh , Evans Philip A. , Raine Andrew 
Published: February 2009

AbstractThe formation of the N‐terminal β‐hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1–17 of ubiquitin folds autonomously and is likely to have a native‐like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1–17)T9D, the native conformation is stabilized with respect to the wild‐type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1–17)T9D peptide does indeed form a hairpin with a native‐like register and a type I turn with a G1 β‐bulge, as in the full‐length protein. The reason for the greater stability of the U(1–17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge‐charge interaction between these residues is responsible.
Position effect of cross‐strand side‐chain interactions on β‐hairpin formation
Authors: Santiveri Clara M. , Rico Manuel , Jiménez M. Angeles 
Published: February 2009

AbstractPrevious conformational analysis of 10‐residue linear peptides enabled us to identify some cross‐strand side‐chain interactions that stabilize β‐hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N‐ and C‐termini of these 10‐residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β‐hairpin formation, we have designed two 15‐residue β‐hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15‐residue peptides suggest that the influence of stabilizing factors for β‐hairpin formation, in particular, cross‐strand side‐chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β‐hairpin folding.
A post‐translational modification of nuclear proteins, NG,NG‐dimethyl‐Arg, found in a natural HLA class I peptide ligand
Authors: Yagüe Jesús , Vázquez Jesús , Castro JOSÉ A. LÓPez De 
Published: February 2009

AbstractPresentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self‐tolerance and induction of cytotoxic T‐cell responses against intracellular parasites. Despite frequent and diverse post‐translational modification of eukaryotic cell proteins, very few class I‐bound peptides with post‐translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA‐B39 (B*3910) derived from an RNA‐binding nucleoprotein that carried NG,NG‐dimethyl‐Arg. Although common among RNA‐binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl‐Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post‐translationally modified ligand was a prominent component (1–2%) of the B*3910‐bound peptide repertoire. The dimethyl‐Arg residue was located in a central position of the peptide, amenable to interacting with T‐cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post‐translationally modified residue may have a major influence on the antigenic properties of this natural ligand.
Automated search of natively folded protein fragments for high‐throughput structure determination in structural genomics
Authors: Kuroda Yutaka , Tani Kazutoshi , Matsuo Yo , Yokoyama Shigeyuki 
Published: February 2009

AbstractStructural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding units (AFU) and have sizes suitable for fast structural analyses. Here, we propose to expand an intuitive procedure often employed for identifying biologically important domains to an automatic method for detecting putative folded protein fragments. The procedure is based on the recognition that large proteins can be regarded as a combination of independent domains conserved among diverse organisms. We thus have developed a program that reorganizes the output of BLAST searches and detects regions with a large number of similar sequences. To automate the detection process, it is reduced to a simple geometrical problem of recognizing rectangular shaped elevations in a graph that plots the number of similar sequences at each residue of a query sequence. We used our program to quantitatively corroborate the premise that segments with conserved sequences correspond to domains that fold into native structures. We applied our program to a test data set composed of 99 amino acid sequences containing 150 segments with structures listed in the Protein Data Bank, and thus known to fold into native structures. Overall, the fragments identified by our program have an almost 50% probability of forming a native structure, and comparable results are observed with sequences containing domain linkers classified in SCOP. Furthermore, we verified that our program identifies AFU in libraries from various organisms, and we found a significant number of AFU candidates for structural analysis, covering an estimated 5 to 20% of the genomic databases. Altogether, these results argue that methods based on sequence similarity can be useful for dissecting large proteins into small autonomously folding domains, and such methods may provide an efficient support to structural genomics projects.
Structural comparison of Ntn‐hydrolases
Authors: Oinonen Carita , Rouvinen Juha 
Published: February 2009

AbstractThe Ntn‐hydrolases (N‐terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N‐terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar αββα‐core structure. The central β‐sheets in the core were found to have different packing angles, ranging from 5 to 35°. In the Ntn‐hydrolases under study, eight totally conserved secondary structure units were found (region C). Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn‐hydrolases. Two additional regions, consisting of secondary structure units (regions A and B), were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.
Presence of closely spaced protein thiols on the surface of mammalian cells
Authors: Donoghue Neil , Yam Patricia T.W. , Jiang Xing‐Mai , Hogg Philip J. 
Published: February 2009

AbstractIt has been proposed that certain cell‐surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane‐impermeable trivalent arsenical to identify closely spaced thiols in cellsurface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4‐(N‐(S‐glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell‐surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the γ‐glutamyl residue of GSAO (GSAO‐B). Incorporation of GSAO‐B into proteins was assessed by measuring the biotin using streptavidin‐peroxidase. Up to 12 distinct proteins were labeled with GSAO‐B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO‐B. These findings demonstrate that the cell‐surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.
Enhancement by Mg2+ of domain specificity in Ca2+‐dependent interactions of calmodulin with target sequences
Authors: Martin Stephen R. , Masino Laura , Bayley Peter M. 
Published: February 2009

AbstractMg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+‐dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+‐binding loops, and the Mg‐calmodulin complex is significantly different from apo‐calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo‐calmodulin. Apo‐calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd ∼ 70‐90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C‐domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+‐saturated calmodulin‐target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+‐dependent target interactions.
Rationale for Bcl‐XL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies
Authors: Petros Andrew M. , Nettesheim David G. , Wang Yi , Olejniczak Edward T. , Meadows Robert P. , Mack Jamey , Swift Kerry , Matayoshi Edmund D. , Zhang Haichao , Fesik Stephen W. , Thompson Craig B. 
Published: February 2009

AbstractThe three‐dimensional structure of the anti‐apoptotic protein Bcl‐XL complexed to a 25‐residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl‐XL bound to a 16‐residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl‐XL. However, based upon site‐directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25‐mer for Bcl‐XL compared to the Bad 16‐mer. Rather, the increased helix propensity of the Bad 25‐mer is primarily responsible for its greater affinity for Bcl‐XL. Based on this observation, a pair of 16‐residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl‐XL. Both peptides showed an increase in helix propensity compared to the wild‐type and exhibited an enhanced affinity for Bcl‐XL.
The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding
Authors: Teplova Marianna , Tereshko Valentina , Egli Martin , Sanishvili Ruslan , Joachimiak Andrzej , Bushueva Tatyana , Anderson Wayne F. 
Published: February 2009

AbstractThe yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation. An example for the latter case is the sua5 gene from yeast. Sua5 was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791‐801). However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene. To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 Å. YrdC adopts a new fold with no obvious similarity to those of other proteins with known three‐dimensional (3D) structure. The protein features a large concave surface on one side that exhibits a positive electrostatic potential. The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein. An investigation of YrdC's binding affinities for single‐ and double‐stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double‐stranded RNA. Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.
The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus
Authors: Thompson Gary S. , Radford Sheena E. , Leung Yun‐Chung , Redfield Christina , Ferguson Stuart J. 
Published: February 2009

AbstractThe solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35 ± 0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double‐wound Greek‐key fold with two α‐helices toward its C‐terminus, similar to that of its oxidized counterpart determined by X‐ray crystallography. Comparison of the Cu(I) solution structure with the X‐ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.
Peptide and metal ion‐dependent association of isolated helix‐loop‐helix calcium binding domains: Studies of thrombic fragments of calmodulin
Authors: Brokx Richard D. , Vogel Hans J. 
Published: February 2009

AbstractCalmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium‐binding regulatory protein, has been cleaved by thrombin to create two fragments, TM1 (1‐106) and TM2 (107‐148). NMR and CD results indicate that TM1 and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix‐loop‐helix domains, but rather within an α‐helix. Cadmium‐113 NMR results show that this complex has enhanced metal‐ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM‐binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl‐13C‐Met‐labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium‐113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM‐dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix‐loop‐helix Ca2+‐binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.