bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal unfolding of tetrameric melittin: Comparison with the molten globule state of cytochrome c
Authors: Hagihara Yoshihisa , Oobatake Motohisa , Goto Yuji
Published: February 2009

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric α‐helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion‐ and pH‐dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid‐denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far‐UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2‐state transition mechanism between the tetrameric α‐helical state and the monomelic unfolded state. From the peptide or salt‐concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)−1 K−1 at 30 °C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)−1 K−1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.
Stabilization of myoglobin by multiple alanine substitutions in helical positions
Authors: Lin Laura , Pinker Rachel J. , Phillips George N. , Kallenbach Neville R.
Published: February 2009

We have carried out a series of multiple Xaa → Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.
The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro
Authors: Guagliardi Annamaria , Cerchia Laura , Bartolucci Simonetta , Rossi Mosé
Published: February 2009

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 °C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920‐kDa oligomer of 57‐kDa subunits, displays a potassium‐dependent ATPase activity with an optimum temperature at 80 °C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride‐denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible in‐activations at 90 °C of several thermophilic enzymes by the binding of the denaturation intermediate; the time‐courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 °C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg‐induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.
The NMR solution structure of the pheromone Er‐2 from the ciliated protozoan Euplotes raikovi
Authors: Ottiger M. , Szyperski T. , Luginbühl P. , Ortenzi C. , Luporini P. , Bradshaw R. A. , WÜthrich K.
Published: February 2009

The NMR structure of the pheromone Er‐2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40‐residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, Cα, and C′ of residues 3–37 was 0.31 Å. The molecular architecture is dominated by an up‐down‐up bundle of 3 short helices of residues 5–11, 14–20, and 23–33, which is similar to the structures of the homologous pheromones Er‐1 and Er‐10. Novel structural features include a well‐defined N‐cap on the first helix, a 1‐residue deletion in the second helix resulting in the formation of a 310‐helix rather than an α‐helix as found in Er‐1 and Er‐10, and the simultaneous presence of 2 different conformations for the C‐terminal tetrapeptide segment, i.e., a major conformation with the Leu 39‐Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.
Derivation of rules for comparative protein modeling from a database of protein structure alignments
Authors: ŠAli Andrej , Overington John P.
Published: February 2009

We describe a database of protein structure alignments as well as methods and tools that use this database to improve comparative protein modeling. The current version of the database contains 105 alignments of similar proteins or protein segments. The database comprises 416 entries, 78,495 residues, 1,233 equivalent entry pairs, and 230,396 pairs of equivalent alignment positions. At present, the main application of the database is to improve comparative modeling by satisfaction of spatial restraints implemented in the program MODELLER (Šali A, Blundell TL, 1993, J Mol Biol 234:779–815). To illustrate the usefulness of the database, the restraints on the conformation of a disulfide bridge provided by an equivalent disulfide bridge in a related structure are derived from the alignments; the prediction success of the disulfide dihedral angle classes is increased to approximately 80%, compared to approximately 55% for modeling that relies on the stereochemistry of disulfide bridges alone. The second example of the use of the database is the derivation of the probability density function for comparative modeling of the cis/trans isomerism of the proline residues; the prediction success is increased from 0% to 82.9% for cis‐proline and from 93.3% to 96.2% for trans‐proline. The database is available via electronic mail.
A neural network model for the prediction of membrane‐spanning amino acid sequences
Authors: Lohmann Reinhard , Schneider Gisbert , Behrens Dirk , Wrede Paul
Published: February 2009

The architecture and weights of an artificial neural network model that predicts putative transmembrane sequences have been developed and optimized by the algorithm of structure evolution. The resulting filter is able to classify membrane/nonmembrane transition regions in sequences of integral human membrane proteins with high accuracy. Similar results have been obtained for both training and test set data, indicating that the network has focused on general features of transmembrane sequences rather than specializing on the training data. Seven physicochemical amino acid properties have been used for sequence encoding. The predictions are compared to hydrophobicity plots.
Viral cell recognition and entry
Authors: Rossmann Michael G.
Published: February 2009

Rhinovirus infection is initiated by the recognition of a specific cell‐surface receptor. The major group of rhino‐virus serotypes attach to intercellular adhesion molecule‐1 (ICAM‐1). The attachment process initiates a series of conformational changes resulting in the loss of genomic RNA from the virion. X‐ray crystallography and sequence comparisons suggested that a deep crevice or canyon is the site on the virus recognized by the cellular receptor molecule. This has now been verified by electron microscopy of human rhinovirus 14 (HRV14) and HRV16 complexed with a soluble component of ICAM‐1.

A hydrophobic pocket underneath the canyon is the site of binding of various hydrophobic drug compounds that can inhibit attachment and uncoating. This pocket is also associated with an unidentified, possibly cellular in origin, “pocket factor.” The pocket factor binding site overlaps the binding site of the receptor. It is suggested that competition between the pocket factor and receptor regulates the conformational changes required for the initiation of the entry of the genomic RNA into the cell.
Sequence determinants of the capping box, a stabilizing motif at the N‐termini of α‐helices
Authors: Seale Jeffrey W. , Srinivasan Rajgopal , Rose George D.
Published: February 2009

The capping box, a recurrent hydrogen bonded motif at the N‐termini of α‐helices, caps 2 of the initial 4 backbone amide hydrogen donors of the helix (Harper ET, Rose GD, 1993, Biochemistry 32:7605–7609). In detail, the side chain of the first helical residue forms a hydrogen bond with the backbone of the fourth helical residue and, reciprocally, the side chain of the fourth residue forms a hydrogen bond with the backbone of the first residue. We now enlarge the earlier definition of this motif to include an accompanying hydrophobic interaction between residues that bracket the capping box sequence on either side. The expanded box motif—in which 2 hydrogen bonds and a hydrophobic interaction are localized within 6 consecutive residues — resembles a glycine‐based capping motif found at helix C‐termini.
Template‐assembled melittin: Structural and functional characterization of a designed, synthetic channel‐forming protein
Authors: Pawlak Michael , Meseth Ulrich , Vogel Horst , Dhanapal Boopathy , Mutter Manfred
Published: February 2009

Template‐assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin‐TASP) or of a truncated melittin sequence (amino acids 6–26, melittin6–26‐TASP), C‐terminally linked to a (linear or cyclic) 10‐amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye‐loaded vesicles. Melittin‐TASP and the truncated analogue preferentially adopt α‐helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin‐TASP self‐aggregates in water. On an air‐water interface, the differently sized molecules can be self‐assembled and compressed to a compact structure with a molecular area of around 600 Å2, compatible with a 4‐helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 × 109 M−1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single‐channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage‐dependent channel activity on membranes of large area. In this respect, melittin‐TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template‐assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.
On the evolution of alternate core packing in eightfold β/α‐barrels
Authors: Raine Andrew R.C. , Scrutton Nigel S. , Mathews F. Scott
Published: February 2009

Two sequence‐related subfamilies of flavin‐binding β/α‐barrels have been identified (the type I and type II proteins) that differ in the nature of residue packing in the core of the barrel domain. Similar observed differences in the packing of internal amino acid side chains in β/α‐barrels have previously been used to argue that these domains have evolved convergently toward a stable structural framework. Using structural alignments of flavin‐binding barrel proteins, we demonstrate that simple genetic alterations may be responsible for switching the nature of side‐chain packing observed in β/α‐barrels. The implication is that the 2 structural classes of β/α‐barrel cores can arise divergently from an ancestral barrel framework and that convergent evolution to a stable fold need not be invoked to account for the emergence of 2 classes of β/α‐barrel core.
The tyrosine corner: A feature of most greek key β‐barrel proteins
Authors: Hemmingsen Jens M. , Gernert Kim M. , Richardson Jane S. , Richardson David C.
Published: February 2009

The Tyr corner is a conformation in which a tyrosine (residue “Y”) near the beginning or end of an antiparallel β‐strand makes an H bond from its side‐chain OH group to the backbone NH and/or CO of residue Y – 3, Y – 4, or Y – 5 in the nearby connection. The most common “classic” case is a Δ4 Tyr corner (more than 40 examples listed), in which the H bond is to residue Y – 4 and the Tyr x1 is near −60°. Y – 2 is almost always a glycine, whose left‐handed β or very extended β conformation helps the backbone curve around the Tyr ring. Residue Y – 3 is in polyproline II conformation (often Pro), and residue Y – 5 is usually a hydrophobic (often Leu) that packs next to the Tyr ring. The consensus sequence, then, is LxPGxY, where the first x (the H‐bonding position) is hydrophilic. Residues Y and Y – 2 both form narrow pairs of β‐sheet H‐bonds with the neighboring strand, Δ5 Tyr corners have a 1‐residue insertion between the Gly and the Tyr, forming a β‐bulge. One protein family has a Δ4 corner formed by a His rather than a Tyr, and several examples use Trp in place of Tyr. For almost all these cases, the protein or domain is a Greek key β‐barrel structure, the Tyr corner ends a Greek key connection, and it is well‐conserved in related proteins. Most low‐twist Greek key β‐barrels have 1 Tyr corner. “Reverse” Δ4 Tyr corners (H bonded to Y + 4) and other variants are described, all less common and less conserved. It seems likely that the more classic Tyr corners (Δ4, Δ5, and Δ3 Tyr, Trp, or His) contribute to the stability of a Greek key connection over a hairpin connection, and also that they may aid in the process of folding up Greek key structures.
Engineering the quaternary structure of an enzyme: Construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli
Authors: Breiter Deborah R. , Resnik Ernesto , Banaszak Leonard J.
Published: February 2009

The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high‐resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh− E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000‐ and 17,500‐fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4‐fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 Å resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.
A structural model for the prostate disease marker, human prostate‐specific antigen
Authors: Villoutreix Bruno O. , Griffin John H. , Getzoff Elizabeth D.
Published: February 2009

Prostate‐specific antigen (PSA) provides an excellent serum marker for prostate cancer, the most frequent form of cancer in American males. PSA is a 237‐residue protease based on sequence homology to kallikrein‐like enzymes. To predict the 3‐dimensional structure of PSA, homology modeling studies were performed based on sequence and structural alignments with tonin, pancreatic kallikrein, chymotrypsin, and trypsin. The structurally conserved regions of the 4 reference X‐ray proteins provided the core structure of PSA, whereas the loop structures were modeled on the loops of tonin and kallikrein. The unique “kallikrein loop” insert, between Ser 95b and Pro 95k of kallikrein, was constructed using molecular mechanics, dynamics, and electrostatics calculations. In the resulting PSA structure, the catalytic triad, involving residues His 57, Asp 102, and Ser 195, and hydrophobic and electrostatic interactions typical of serine proteases were extremely well conserved. Similarly, the 5 disulfide bonds of kallikrein were also conserved in PSA. These results, together with the fact that no major steric clashes arose during the modeling process, provide strong evidence for the validity of the PSA model. Calculation of the electrostatic potential contours of kallikrein and PSA was carried out using the finite difference Poisson‐Boltzmann method. The calculations revealed matching areas of negative potential near the catalytic triad, but differences in the positive potential surrounding the active site. The PSA glycosylation site, Asn 61, is fully accessible to the solvent and is enclosed in a positive region of the isopotential map. The bottom of the substrate specificity pocket, residue SI, is a serine (Ser 189) as in chymotrypsin, rather than aspartate (Asp 189) as in tonin, kallikrein, and trypsin. This fact, plus other features of the SI binding‐pocket region, suggest that PSA would prefer substrates with hydrophobic residues at the PI position. The location of a potential zinc ion binding site involving the side chain of histidines 91, 101, and 233 is also suggested. This PSA model should facilitate the understanding and prediction of structural and functional properties of this important cancer marker.
Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling
Authors: Koonin Eugene V. , Tatusov Roman L. , Altschul Stephen F. , Bryant Stephen H. , Mushegian Arcady R. , Bork Peer , Valencia Alfonso
Published: February 2009

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N‐terminal domain related to class θ glutathione S‐transferases (GST). GST‐like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress‐induced proteins from bacteria and plants, bacterial reductive dehalogenases and β‐etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST‐related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST‐like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl‐tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.
Unique dicistronic operon (ptsI‐crr) in Mycoplasma capricolum encoding Enzyme I and the glucose‐specific Enzyme IIA of the phosphoenolpyruvate: Sugar phosphotransferase system: Cloning, sequencing, promoter analysis, and protein characterization
Authors: Zhu Peng‐Peng , Peterkofsky Alan , Reizer Jonathan
Published: February 2009

The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the pts operon. In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS. In M. capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531–26540). In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc. Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene. Primer extension studies established the transcription start sites for the ptsI and crr genes. The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram‐positive than to those from gram‐negative organisms. The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids. The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue. In vitro sugar phosphorylation studies with extracts from E. coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E. coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E. coli and B. subtilis PTS components.
A structural comparison of 21 inhibitor complexes of the aspartic proteinase from Endothia parasitica
Authors: Bailey David , Cooper Jonathan B.
Published: February 2009

The aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E), and AIDS (HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X‐ray analyses have shown that transition‐state isostere inhibitors of aspartic proteinases bind in similar extended conformations in the active‐site cleft of the target enzyme. Upon comparison of 21 endothiapepsin inhibitor complexes, the hydrogen bond lengths were found to be shortest where the isostere (P1–P'1) interacts with the enzyme's catalytic aspartate pair. Hydrogen bonds with good geometry also occur at P'2, and more so at P3, where a conserved water molecule is involved in the interactions. Weaker interactions also occur at P2, where the side‐chain conformations of the inhibitors appear to be more variable than at the more tightly held positions. At P2 and, to a lesser extent, P3, the side‐chain conformations depend intriguingly on interactions with spatially adjacent inhibitor side chains, namely P'1 and P1, respectively. The tight binding at P1–P'1, P3, and P'2 is also reflected in the larger number of van der Waals contacts and the large decreases in solvent‐accessible area at these positions, as well as their low temperature factors. Our analysis substantiates earlier proposals for the locations of protons in the transition‐state complex. Aspartate 32 is probably ionized in the complexes, its charge being stabilized by 1, or sometimes 2, hydrogen bonds from the transition‐state analogues at P1. The detailed comparison also indicates that the P1 and P2 residues of substrate in the ES complex may be strained by the extensive binding interactions at P3, P'1, and P'2 in a manner that would facilitate hydrolysis of the scissile peptide bond.
Immunoglobulin fold characteristics of B7–1 (CD80) and B7–2 (CD86)
Authors: Bajorath Jürgen , Peach Robert J. , Linsley Peter S.
Published: February 2009

B7–1 and B7–2 are expressed on antigen‐presenting cells and bind to the CD28 and CTLA‐4 receptors on T cells. These interactions trigger a costimulatory pathway that is essential for T‐cell activation. B7–1 and B7–2 are members of the immunoglobulin superfamily (IgSF) and, despite sharing common function, have only limited sequence similarity. The B7–1 extracellular region was previously subdivided into 2 IgSF domains, an N‐terminal V(ariable)‐like domain, followed by a C(onstant)‐like domain. We recently reported that the V‐like domains of B7–1 and B7–2 share some significant sequence similarities with 3 major histocompatibility complex (MHC)‐encoded members of the IgSF. We have now applied inverse folding methodology to assess the compatibility of the B7–1 and B7–2 extracellular region sequences with currently available 3‐dimensional structures. In these calculations, the sequences of the N‐terminal (V‐like) domains in B7–1 and B7–2 were not compatible with known structures, including the IgSF V‐set. In contrast, the sequences of the C‐like domains were compatible with IgSF C‐set structures and were best recognized by the β2‐microglobulin (β2m) domain of MHC Class I. A sequence comparison of the C‐like domains in the B7 molecules showed that 11 of 17 rigorously conserved residues in B7–1 and B7–2 are not IgSF C‐1 set consensus residues. When mapped onto the corresponding positions of the β2m structure, the conserved residues in B7 cluster on the surface, where they may interact with the B7 V‐like domain or other molecules.
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

In 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

The 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

Interleukin‐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

By 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

Inteins (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

Measurement 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

We 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

We 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

The 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

A 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

We 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.