bluuebunny/crossref_metadata_2025_split huggingface dataset:
Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in λ repressor
Authors: Marqusee Susan , Sauer Robert T. 
Published: February 2009

AbstractIn the N‐terminal domain of λ repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild‐type N‐terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side‐chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild‐type side chain on protein stability have been estimated. These studies indicate that the Asp 14–Arg 17 and Asp 14–Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of α‐helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.
On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase
Authors: Schendel Sharon L. , Cramer William A. 
Published: February 2009

AbstractThe transition of the colicin E1 channel polypeptide from a water‐soluble to membrane‐bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a “molten globule,” or “compact unfolded,” intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel‐induced solute efflux and the amplitude of the near‐UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near‐UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near‐UV CD spectrum, are retained over the pH range 3–4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2–6. The temperature independence of the near‐UV spectrum at pH 3–6 up to 70°C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58°C in the amplitude of the near‐UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a “compact unfolded” state, having some, but not all of the properties of a “molten globule.”The small effect on local membrane acidity of a physiological acidic membrane lipid content, the retention of significant near‐UV CD amplitude down to pH 2, and the small extent of immersion of the 40‐Å globular colicin channel polypeptide in the 10‐Å lower pH layer at the membrane surface make it unlikely that a local lower pH at the membrane surface significantly facilitates formation of an unfolded intermediate.
Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding
Authors: Hammacher Annet , Ward Larry D. , Simpson Richard J. , Weinstock Janet , Treutlein Herbert , Yasukawa Kiyoshi 
Published: February 2009

AbstractInterleukin‐6 (IL‐6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL‐6 may fold as a 4 α‐helix bundle structure with up‐up‐down‐down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL‐6 polypeptides display distinct species‐specific activities. Although human IL‐6 (hIL‐6) is active in both human and mouse cell assays, mouse IL‐6 (mIL‐6) is not active on human cells. Previously, we demonstrated that the 5 C‐terminal residues of mIL‐6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472–1481). To further probe the structure‐function relationship of this cytokine, we have constructed several human/mouse IL‐6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu‐62 (Lys‐65 in mIL‐6) in the putative connecting loop AB between helices A and B, at Arg‐113 (Val‐117 in mIL‐6) at the N‐terminal end of helix C, at Lys‐150 (Asp‐152 in mIL‐6) in the connecting loop CD between helices C and D, and at Leu‐178 (Thr‐180 in mIL‐6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity.The conformational integrity of the IL‐6 hybrids was assessed by far‐UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL‐6, residues 178–184 in helix D and residues 63–113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL‐6 receptor. For human IL‐6, it would appear that interactions between residues Ala‐180, Leu‐181, and Met‐184 and residues in the N‐terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL‐6 correlated with a significant loss of α‐helical content and a 200‐fold reduction in activity in the mouse bioassay. A homology model of mIL‐6 based on the X‐ray structure of human granulocyte colony‐stimulating factor is presented.
Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sites
Authors: Wu Jianhua , Frillingos Stathis , Voss John , Ronald Kaback H. 
Published: February 2009

AbstractBy using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right‐side‐out membrane vesicles containing Val 331 → Cys permease, lactose transport is inactivated by either N‐ethylmaleimide (NEM) or 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin (CPM). Remarkably, β,d‐galactopyranosyl 1‐thio‐β,d‐galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331 → Cys permease was then purified and studied in dodecyl‐β,d‐maltoside by site‐directed fluorescence spectroscopy. The reactivity of Val 331 → Cys permease with 2‐(4′‐maleimidylanilino)‐naphthalene‐6‐sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (<0.8 mM), but the fluorescence of the MIANS‐labeled protein is quenched in a saturable manner (apparent Kd ≌ 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1–10 mM), the reactivity of Val 331 → Cys permease with MIANS is enhanced and the emission maximum of MIANS‐labeled permease is blue shifted by 3–7 nm. Furthermore, the fluorescence of MIANS‐labeled Val 331 → Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%. Finally, the EPR spectrum of nitroxide spin‐labeled Val 331 → Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS‐labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.
Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins
Authors: Pietrokovski Shmuel 
Published: February 2009

AbstractInteins (protein introns) are internal portions of protein sequences that are posttranslationally excised while the flanking regions are spliced together, making an additional protein product. Inteins have been found in a number of homologous genes in yeast, mycobacteria, and extreme thermophile archaebacteria. The inteins are probably multifunctional, autocatalyzing their own splicing, and some were also shown to be DNA endonucleases. The splice junction regions and two regions similar to homing endonucleases were thought to be the only common sequence features of inteins.This work analyzed all published intein sequences with recently developed methods for detecting weak, conserved sequence features. The methods complemented each other in the identification and assessment of several patterns characterizing the intein sequences. New intein conserved features are discovered and the known ones are quantitatively described and localized. The general sequence description of all the known inteins is derived from the motifs and their relative positions. The intein sequence description is used to search the sequence databases for intein‐like proteins. A sequence region in a mycobacterial open reading frame possessing all of the intein motifs and absent from sequences homologous to both of its flanking sequences is identified as an intein. A newly discovered putative intein in red algae chloroplasts is found not to contain the endonuclease motifs present in all other inteins. The yeast HO endonuclease is found to have an overall intein‐like structure and a few viral polyprotein cleavage sites are found to be significantly similar to the inteins amino‐end splice junction motif. The intein features described may serve for detection of intein sequences.
Mass spectrometric measurement of protein amide hydrogen exchange rates of apo‐ and holo‐myoglobin
Authors: Johnson Richard S. , Walsh Kenneth A. 
Published: February 2009

AbstractMeasurement of backbone amide hydrogen exchange rates can provide detailed information concerning protein structure, dynamics, and interactions. Although nuclear magnetic resonance is typically used to provide these data, its use is restricted to lower molecular weight proteins that are soluble at millimolar concentrations. Not subject to these limitations is a mass spectrometric approach for measuring deuterium incorporation into proteins that are subsequently proteolyzed by pepsin; the resulting peptide masses are measured using a flowing‐fast atom bombardment ionization source (Zhang Z, Smith DL, 1993, Protein Sci 2:522–531). In the current study, amide deuterium incorporation for intact apo‐ and holo‐myoglobin was measured using liquid chromatography coupled directly to an electrospray ionization (LC/MS) source. Electrospray ionization provided a more complete coverage of the protein sequence and permitted the measurement of deuterium incorporation into intact proteins. Tandem mass spectrometry was used to rapidly identify the peptic peptides. It was found that within 30 s, the amides in apo‐myoglobin were 47% deuterated, whereas holo‐myoglobin was 12% deuterated. Peptic digestion and LC/MS demonstrated that regions represented by peptic peptides encompassing positions 1–7, 12–29, and 110–134 were not significantly altered by removal of the heme. Likewise, destabilized regions were identified within positions 33–106 and 138–153.
Modeling of the spatial structure of eukaryotic ornithine decarboxylases
Authors: Grishin Nick V. , Phillips Margaret A. , Goldsmith Elizabeth J. 
Published: February 2009

AbstractWe used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate‐utilizing enzymes. Our studies suggest that the N‐terminal domain of ornithine decarboxylase folds into a β/α‐barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate‐binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine‐rich sequence both map to the C‐termini of the β‐strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.
N‐ and C‐capping preferences for all 20 amino acids in α‐helical peptides
Authors: Doig Andrew J. , Baldwin Robert L. 
Published: February 2009

AbstractWe have determined the N‐ and C‐capping preferences of all 20 amino acids by substituting residue X in the peptides NH2‐XAKAAAAKAAAAKAAGY‐CONH2 and in Ac‐YGAAKAAAAKAAAAKAX‐CO2H. Helix contents were measured by CD spectroscopy to obtain rank orders of capping preferences. The data were further analyzed by our modified Lifson‐Roig helix‐coil theory, which includes capping parameters (n and c), to find free energies of capping (‐RT In n and —RT In c), relative to Ala. Results were obtained for charged and uncharged termini and for different charged states of titratable side chains. N‐cap preferences varied from Asn (best) to Gin (worst). We find, as expected, that amino acids that can accept hydrogen bonds from otherwise free backbone NH groups, such as Asn, Asp, Ser, Thr, and Cys generally have the highest N‐cap preference. Gly and the acetyl group are favored, as are negative charges in side chains and at the N‐terminus. Our N‐cap preference scale agrees well with preferences in proteins. In contrast, we find little variation when changing the identity of the C‐cap residue. We find no preference for Gly at the C‐cap in contrast to the situation in proteins. Both N‐cap and C‐cap results for Tyr and Trp are inaccurate because their aromatic groups affect the CD spectrum. The data presented here are of value in rationalizing mutations at capping sites in proteins and in predicting the helix contents of peptides.
A sequence‐coupled vector‐projection model for predicting the specificity of GalNAc‐transferase
Authors: Chou Kuo‐Chen 
Published: February 2009

AbstractThe specificity of GalNAc‐transferase is consistent with the existence of an extended site composed of nine sub‐sites, denoted by R4, R3, R2, Rb R0, R1, R2′, R3′, and R4, where the acceptor at R0 is either Ser or Thr to which the reducing monosaccharide is being anchored. To predict whether a peptide will react with the enzyme to form a Ser‐ or Thr‐conjugated glycopeptide, a new method has been proposed based on the vector‐projection approach as well as the sequence‐coupled principle. By incorporating the sequence‐coupled effect among the subsites, the interaction mechanism among subsites during glycosylation can be reflected and, by using the vector projection approach, arbitrary assignment for insufficient experimental data can be avoided. The very high ratio of correct predictions versus total predictions for the data in both the training and the testing sets indicates that the method is self‐consistent and efficient. It provides a rapid means for predicting O‐glycosylation and designing effective inhibitors of GalNAc‐transferase, which might be useful for targeting drugs to specific sites in the body and for enzyme replacement therapy for the treatment of genetic disorders.
A new computational model for protein folding based on atomic solvation
Authors: Wang Yuhong , Zhang Hui , Scott Robert A. 
Published: February 2009

AbstractA new model for calculating the solvation energy of proteins is developed and tested for its ability to identify the native conformation as the global energy minimum among a group of thousands of computationally generated compact non‐native conformations for a series of globular proteins. In the model (called the WZS model), solvation preferences for a set of 17 chemically derived molecular fragments of the 20 amino acids are learned by a training algorithm based on maximizing the solvation energy difference between native and non‐native conformations for a training set of proteins. The performance of the WZS model confirms the success of this learning approach; the WZS model misrecognizes (as more stable than native) only 7 of 8,200 non‐native structures. Possible applications of this model to the prediction of protein structure from sequence are discussed.
Modeling protein loops using a ϕi+1, Ψi dimer database
Authors: Sudarsanam Sucha , Dubose Robert F. , March Carl J. , Srinivasan Subhashini 
Published: February 2009

AbstractWe present an automated method for modeling backbones of protein loops. The method samples a database of ϕi+1 and Ψi angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles ϕi+1 and Ψi completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed ϕi+1, Ψi pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N‐terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of ϕi+1, Ψi for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three‐dimensional structures. Typically, 10,000–50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50–80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near‐native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near‐native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop‐closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near‐native loop conformations.
Volume changes of the molten globule transitions of horse heart ferricytochrome c: A thermodynamic cycle
Authors: Foygel Kira , Spector Shari , Chatterjee Sukalyan , Kahn Peter C. 
Published: February 2009

AbstractVolume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be —136 ± 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded +100 ± 6 mL/mol. MG to N was broken into two steps, N to NClX at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClX = N interacting with an unknown number, X, of chloride ions), and MG to NClX by jumping MG at pH 2 in 0.5 M KCl to pH 7 at the same salt concentration. The ΔV of N to NClX was —30.9 ± 1.4 mL/mol protein, whereas MG to NClX entailed a ΔV of —235 ± 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.
Structural analysis of the N‐ and C‐termini in a peptide with consensus sequence
Authors: Gong Youxiang , Zhou Hongxing X. , Guo Mingming , Kallenbach Neville R. 
Published: February 2009

AbstractWe present a structural analysis of a peptide, the sequence of which includes amino acids that show preferences for specific positions near the N‐ and C‐termini in protein helices. This peptide has the sequence ac‐YMSEDELKAAEAAFKRHGVP‐amide, which includes a strong version of an N‐terminal Harper‐Rose capping box structure as well as a Gly located close to the C‐terminus designed to elucidate its role in C‐terminal capping. The sequence of five residues at the middle is inserted to separate effects at the two ends via a helix‐stabilizing linker. Application of a simulated annealing procedure using interproton distance constraints derived from 1H NOESY experiments in water reveals the presence of a C‐terminal structure in this model. The C‐terminus forms a folded back structure in a significant fraction of structures generated by the annealing, in most of which Gly assumes an αL conformation. This structure occurs within a highly flexible region of the molecule and hence is occupied only a fraction of the time.
Hints on the evolutionary design of a dimeric RNase with special bioactions
Authors: Donato Alberto Di , Cafaro Valeria , Romeo Ivana , D'Alessio Giuseppe 
Published: February 2009

AbstractResidues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394–17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N‐terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N‐terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N‐teminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.