bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of antibody affinity by a non‐contact residue
Authors: Schildbach Joel F. , Near Richard I. , Bruccoleri Robert E. , Haber Edgar , Jeffrey Philip D. , Novotny Jiri , Sheriff Steven , Margolies Michael N.
Published: July 2010

Antibody LB4, produced by a spontaneous variant of the murine anti‐digoxin monoclonal antibody 26–10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640–4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26–10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26–10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity. The mutagenesis and modeling results suggest that even conservative replacements of non‐contact residues can alter affinity indirectly through their impact on contact residue placement.
A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways
Authors: Wilkins James A. , Cone James , Randhawa Zafar I. , Wood David , Warren M. Kim , Witkowska H. Ewa
Published: July 2010

The folding pathway for a 150‐amino acid recombinant form of the dimeric cytokine human macrophage colony‐stimulating factor (M‐CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non‐native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M‐CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell‐based assay. The results presented here strongly suggest that M‐CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Mannose‐specific lectins bind α‐2‐macroglobulin and an unknown protein from human plasma
Authors: Van Leuven F. , Torrekens S. , Van Berghe H. Den , Van Damme E. , Peumans W.
Published: July 2010

GNA, the mannose‐specific lectin from Galanthus nivalis was confirmed to bind α‐2‐macroglobulin (A2M) but another protein was copurified with A2M from total human plasma. A total of 23 other lectins with diverse specificities were tested for reaction with human A2M and with three other members of the A2M family. NPA, a mannose‐specific lectin isolated from Narcissus pseudonarcissus bulbs, and RSA, the Rhizoctonia solani agglutinin, were selected for further testing. For isolation of A2M, immobilized NPA was superior to GNA because its binding capacity was an order of magnitude higher. The specificity of these lectins must be very similar however, because the same unknown plasma protein was also bound by NPA. A2M and the unknown protein must share a unique mannose carbohydrate structure not present in any other human plasma protein. The copurified protein subunit size of 185 kDa is very similar to that of A2M, but the native molecular mass of 350 kDa indicated a noncovalent homodimer structure. Together with the acid isoelectric point this is not typical for any known plasma protein nor for any unidentified spot on the two‐dimensional map of human plasma proteins. No immunological reaction with available antisera was evident. A specific antiserum raised to the unknown protein demonstrated its presence in all human plasma samples examined. The N‐terminal residue was blocked, whereas internal protein sequences obtained after CNBr fragmentation and proteolysis were not homologous to any known protein sequence. These data demonstrate that this protein is unknown and not a proteinase inhibitor of the A2M family.
Relationship between sequence conservation and three‐dimensional structure in a large family of esterases, lipases, and related proteins
Authors: Cygler Miroslaw , Schrag Joseph D. , Sussman Joel L. , Harel Michal , Silman Israel , Gentry Mary K. , Doctor Bhupendra P.
Published: July 2010

Based on the recently determined X‐ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three‐dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X‐ray structures.
Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers
Authors: Zhu Bing‐Yan , Zhou Man E. , Kay Cyril M. , Hodges Robert S.
Published: July 2010

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three‐dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA‐binding proteins (leucine zippers), Leu residues are conserved at positions “d,” and β‐branched amino acids, Ile and Val, often occur at positions “a” in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions “a” (9, 16, and 23) or “d” (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn·l. The results of the mutations of Ile to Val at either positions “a” or “d” in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (δδG values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions “a” in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (δδG values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic α‐helices. These structural findings correlate with the observation that in DNA‐binding proteins, the conserved Leu residues at positions “d” are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions “a.”
Structure of recombinant ricin A chain at 2.3 Å
Authors: Mlsna Debra , Monzingo Arthur F. , Katzin Betsy J. , Ernst Stephen , Robertus Jon D.
Published: July 2010

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N‐glycosidase. RTA must be separated from B chain to attack rRNA. The X‐ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild‐type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site‐directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136–1145).
Peptidylglycine α‐amidating monooxygenase: A multifunctional protein with catalytic, processing, and routing domains
Authors: Eipper Betty A. , Milgram Sharon L. , Jean Husten E. , Yun Hye‐Young , Mains Richard E.
Published: July 2010

Peptide α‐amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine α‐amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final α‐amidated product peptide. Tissue‐specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue‐specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH‐terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue‐specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendo‐crine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.
Sequence analyses and evolutionary relationships among the energy‐coupling proteins enzyme I and HPr of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system
Authors: Reizer Jonathan , Hoischen Christian , Reizer Aiala , Pham Tam N. , Saier Milton H.
Published: July 2010

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158–9169). We now report the sequencing of the ptsl gene of B. subtilis encoding Enzyme I (570 amino acids and 63, 076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source‐dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate:phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequenced HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross‐reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N‐terminal and the C‐terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms. Quantitative analysis of this bias indicated that the ptsH genes have higher bias toward codons favored in highly expressed genes than the ptsI genes, in agreement with their relative levels of expression.
Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated IIIGlc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques
Authors: Pelton J.G. , Torchia D.A. , Meadow N.D. , Roseman S.
Published: July 2010

IIIGlc is an 18.1‐kDa signal‐transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two‐dimensional 1H‐15N and 1H‐13C heteronuclear multiple‐quantum coherence (HMQC) experiments and a two‐dimensional 13C‐13C‐1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N‐labeled and uniformly 15N/13C‐labeled protein samples. The experiments rely on one‐bond and two‐bond J couplings that allowed for assignment of the signals without the need for the analysis of through‐space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His‐75 exists predominantly in the Nε2‐H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His‐90 exists primarily in the Nδ1‐H state in the unphosphorylated protein. Upon phosphorylation of the Nε2 nitrogen of His‐90, the Nδ1 nitrogen remains protonated, resulting in the formation of a charged phospho‐His‐90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pKa values for both His‐75 and His‐90 in IIIGlc and His‐75 in phospho‐IIIGlc are less than 5.0, and that the pKa value for phospho‐His‐90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.
Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry
Authors: Tanaka Akiyoshi , Sturtevant Julian M. , Flanagan John
Published: July 2010

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, ΔΔGd0, produced by the mutations in the pH range 5.0–7.0 varied from 1.9 kcal mol−1 (apparent stabilization) for H124L to ‐2.8 kcal mol−1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between ΔΔGd0 and the corresponding values for ΔΔHd and TΔΔSd0, the latter quantities being in most cases much larger in magnitude than ΔΔGd0. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X‐ray crystallography.
Crevice‐forming mutants in the rigid core of bovine pancreatic trypsin inhibitor: Crystal structures of F22A, Y23A, N43G, and F45A
Authors: Danishefsky Avis T. , Housset Dominique , Wlodawer Alexander , Kim Key‐Sun , Tao Feng , Fuchs James , Woodward Clare
Published: July 2010

Crystal structures of four mutants of bovine pancreatic trypsin inhibitor (F22A, Y23A, N43G, and F45A), engineered to alter their stability properties, have been determined. The mutated residues, which are highly conserved among Kunitz‐type inhibitors, are located in the rigid core of the molecule. Replacement of the partially buried bulky residues of the wild‐type protein with smaller residues resulted in crevices open to the exterior of the molecule. The overall three‐dimensional structure of these mutants is very similar to that of the wild‐type protein and only small rearrangements are observed among the atoms lining the crevices.
Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation
Authors: Hazes Bart , Kalk Kor H. , Hol WIM G.J. , Magnus Karen A. , Bonaventura Celia , Bonaventura Joseph , Dauter Zbigniew
Published: July 2010

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 Å. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 Å between both copper ions in the oxygen‐binding site. Each copper has approximate trigonal planar coordination by three histidine Nε atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis‐peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the Nδ atom of the copper B ligand His 324; (2) localization of a chloride‐binding site in the interface between the first and second domain; (3) localization of a putative calcium‐binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5° with respect to the other two domains, whereas within the hexamer the major movement is a 3.1° rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe 49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.
A model for the calmodulin—peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin—myosin light chain kinase peptide complex
Authors: Sekharudu C.Y. , Sundaralingam M.
Published: July 2010

In the crystal structure of troponin C, the holo C‐domain is bound in a head‐to‐tail fashion to the A‐helix of the apo N‐domain of a symmetry‐related molecule. Using this interaction, we have proposed a model for the calmodulin‐peptide complex. We find that the interaction of the C‐domain with the A‐helix is similar to that observed in the NMR structure of the calmodulin‐myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin‐binding cleft and to rationalize the amino acid sequence‐dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234–248, 1990) in that the peptides are rotated by 100° in the calmodulin binding cleft.
Investigations of the thermostability of rubredoxin models using molecular dynamics simulations
Authors: Bradley Elizabeth A. , Adams MICHAEL W.W. , Wampler John E. , Stewart David E.
Published: July 2010

The affects of differences in amino acid sequence on the temperature stability of the three‐dimensional structure of the small β‐sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100°C, were compared to three mesophilic Rds of known X‐ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature‐dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature‐dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.
Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides
Authors: Weng G. , Chen C.X. , Balogh‐Nair V. , Callender R. , Manor D.
Published: July 2010

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF‐Tu (elongation factor Tu from Escherichia coli) and the c‐Harvey ras protein, p21 (the gene product of the human c‐H‐rus proto‐oncogene). Raman spectra of proteins complexed with GDP (guanosine 5′ diphosphate), IDP (inosine 5′ diphosphate), 6‐thio‐GDP, and 6‐18O‐GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6‐keto and 2‐amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (δH) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF‐Tu. Despite the structural similarity of the binding sites of EF‐Tu and p21, the strengths of the observed hydrogen bonds at the 6‐keto and 2‐amino positions vary substantially, by up to a factor of 2. The hydrogen bond between the protein and the 6‐keto group is stronger for GDP in EF‐Tu compared to p21, but this comparison is reversed for bound IDP. Thus, the removal of the 2‐amino group from GDP to form IDP affects binding properties distal to the amino site.
Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 Å resolution
Authors: Kreusch A. , Neubüser A. , Schiltz E. , Weckesser J. , Schulz G.E.
Published: July 2010

The crystal structure of a membrane channel, homotrimeric porin from Rhodopseudomonas blastica has been determined at 2.0 Å resolution by multiple isomorphous replacement and structural refinement. The current model has an R‐factor of 16.5% and consists of 289 amino acids, 238 water molecules, and 3 detergent molecules per subunit. The partial protein sequence and subsequently the complete DNA sequence were determined. The general architecture is similar to those of the structurally known porins. As a particular feature there are 3 adjacent binding sites for n‐alkyl chains at the molecular 3‐fold axis. The side chain arrangement in the channel indicates a transverse electric field across each of the 3 pore eyelets, which may explain the discrimination against nonpolar solutes. Moreover, there are 2 significantly ordered girdles of aromatic residues at the nonpolar/polar borderlines of the interface between protein and membrane. Possibly, these residues shield the polypeptide conformation against adverse membrane fluctuations.
Solution structure of villin 14T, a domain conserved among actin‐severing proteins
Authors: Markus Michelle A. , Nakayama Tomoko , Matsudaira Paul , Wagner Gerhard
Published: July 2010

The solution structure of the N‐terminal domain of the actin‐severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin‐severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N‐terminal domain of villin (14T) contains a central β‐sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2‐stranded parallel β‐sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N‐terminal end of the longest helix, with a Kd of 1.8 ± 0.4 mM, and a weaker site near the C‐terminal end of the same helix, with a Kd of 11 ± 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central β‐sheet.
Two conformational states of Candida rugosa lipase
Authors: Grochulski Pawel , Li Yunge , Schrag Joseph D. , Cygler Miroslaw
Published: July 2010

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 Å resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain — the flap. Comparison of this structure with the previously determined “open” form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the “open” conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis‐trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.
Positions of His‐64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors
Authors: Smith Graham M. , Alexander Richard S , Christianson David W. , Mckeever Brian M. , Ponticello Gerald S. , Springer James P. , Randall William C. , Baldwin John J. , Habecker Charles N.
Published: July 2010

The 3‐dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, la, lb, and lc, has been determined by X‐ray crystallographic methods. The 3 inhibitors (la = C8H12N2O4S3) vary only in the length of the substituent on the 4‐amino group: la, proton; lb, methyl; and lc, ethyl. The binding constants (Ki's) for la, Ib, and lc to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors la and lb, electron density can be observed for His‐64 and a bound water molecule in the native positions. When inhibitor lc is bound, the side chain attached to the 4‐amino group is positioned so that His‐64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason lc binds tighter to HCAII than does la or lb appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His‐64 side chain in a higher energy state.
Studies on the specificity of acetylaminoacylpeptide hydrolase
Authors: Sokolik Charles W. , Liang T. Chyau , Wold Finn
Published: July 2010

In a continuing attempt to explore the types of specificity determinants that may affect protein‐protein (peptide) interactions, a number of short (2‐5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl‐peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac‐AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat, for the different substrates were determined by standard steady‐state kinetics, and the corresponding δδGT D̊x value derived from kcat/Km was used for the comparison, setting δδGT D̊ for Ac‐AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1′, 2′, or 3′ (δδGT D̊ values of 2‐5 kJ); the negative charge provided by the C‐terminus of the substrate also appears to be important, since the amide and O‐Me ester derivatives caused a change in δδGT D̊ values of ‐7 to ‐8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582‐589), and the proposed active site model incorporates subsites for both charge‐charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make‐up of each peptide and not only the effect of a single residue in a given position. Thus, while the peptides with single Asp or Phe substitutions in 1′, 2′, and 3′ gave δδGT D̊ values of 3‐5 kJ, the peptide containing all 3 modifications, Ac‐ADDF, gave only 1 kJ. Similarly, Ac‐TAAA was a poor substrate and Ac‐GAAA was not cleaved at all in this study, while in the past other peptides such as Ac‐TGG and Ac‐GGG have been found to be excellent and reasonably good substrates, respectively. Although the rate differences observed in this work are minor, they nevertheless appear to reflect the kind of structural detail that is involved in determining the specificity of protein‐protein (peptide) interactions.
The native state of apomyoglobin described by proton NMR spectroscopy: Interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS
Authors: Cocco Melanie J. , Lecomte Juliette T.J.
Published: July 2010

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐l‐oxy (HyTEMPO) and the fluorescent dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His‐64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His‐82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481‐6491). The packing of the side chains and the low pKa of His‐64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter‐amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.
Enzyme IIB bcellobiose of the phosphoenol‐pyruvate‐dependent phosphotransferase system of Escherichia coli: Backbone assignment and secondary structure determined by three‐dimensional NMR spectroscopy
Authors: Ab Eiso , Schuurman‐Wolters Gea K. , Saier Milton H. , Reizer Jonathan , Jacuinod Michel , Roepstorff Peter , Dijkstra Klaas , Scheek Ruud M. , Robillard George T.
Published: July 2010

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose‐specific phosphoenol‐pyruvate‐dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13Cα resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H‐NOE 1H‐15N single quantum coherence (15N‐NOESY‐HSQC) spectroscopy and heteronuclear 1H total correlation 1H‐15N single quantum coherence (15N‐TOCSY‐HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13Cα and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3‐dimensional 1Ha‐13Cα‐13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1Hα and 13Cα chemical shifts from their random coil values and 1H‐NOE information from the 15N‐NOESY‐HSQC. These data show that enzyme IIBcellobiose consists of a 4‐stranded parallel β‐sheet and 5 α‐helices. In the wild‐type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a β‐strand.
Characterization of a truncated form of arrestin isolated from bovine rod outer segments
Authors: Palczewski Krzysztof , Buczylko Janina , Ohguro Hiroshi , Annan Roland S. , Carr Steven A. , Crabb John W. , Kaplan Michael W. , Johnson Richard S. , Walsh Kenneth A.
Published: July 2010

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48‐kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44‐kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C‐terminal 35 residues (positions 370‐404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N‐ and C‐termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.
Sequence relationships between integral inner membrane proteins of binding protein‐dependent transport systems: Evolution by recurrent gene duplications
Authors: Saurin W. , Dassa E.
Published: July 2010

Periplasmic binding protein‐dependent transport systems are composed of a periplasmic substrate‐binding protein, a set of 2 (sometimes 1) very hydrophobic integral membrane proteins, and 1 (sometimes 2) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters. We performed a molecular phylogenetic analysis of the sequences of 70 hydrophobic membrane proteins of these transport systems in order to investigate their evolutionary history. Proteins were grouped into 8 clusters. Within each cluster, protein sequences displayed significant similarities, suggesting that they derive from a common ancestor. Most clusters contained proteins from systems transporting analogous substrates such as monosaccharides, oligopeptides, or hydrophobic amino acids, but this was not a general rule. Proteins from diverse bacteria are found within each cluster, suggesting that the ancestors of current clusters were present before the divergence of bacterial groups. The phylogenetic trees computed for hydrophobic membrane proteins of these per‐meases are similar to those described for the periplasmic substrate‐binding proteins. This result suggests that the genetic regions encoding binding protein‐dependent permeases evolved as whole units. Based on the results of the classification of the proteins and on the reconstructed phylogenetic trees, we propose an evolutionary scheme for periplasmic permeases. According to this model, it is probable that these transport systems derive from an ancestral system having only 1 hydrophobic membrane protein. None of the proteins considered in this study display detectable sequence similarity to hydrophobic membrane proteins or domains from other ABC transporters such as bacterial polysaccharide export systems, bacterial toxin proteins exporters, and eukaryotic ABC proteins. It is likely that they constitute a specific subfamily within the superfamily of ABC transporters.
Rational design of a three‐heptad coiled‐coil protein and comparison by molecular dynamics simulation with the GCN4 coiled coil: Presence of interior three‐center hydrogen bonds
Authors: Rozzelle James E. , Tropsha Alexander , Erickson Bruce W.
Published: July 2010

α‐Helical coiled coils have a 7‐residue repeating pattern (abcdefg) where a and d are usually hydrophobic. We have designed a 2‐stranded 44‐residue coiled‐coil protein (P44) consisting of 222‐residue α‐helices linked by 2 terminal disulfide groups to test whether the disulfide bridges could stabilize a 3‐heptad coiled coil. P44 should be stabilized by intrahelical hydrogen bonds, interhelical disulfide and salt bridges, and interior hydrophobic interactions. A computer model of P44 was built and its stability was studied by molecular dynamics simulation with explicit water. This doubly crosslinked 3‐heptad coiled coil did not unfold during a 300‐ps simulation. But reduced P44 with 4 thiol groups did unfold. For comparison, the 62‐residue crystal structure of the 4‐heptad coiled coil of transcription activator GCN4 did not unfold during a 300‐ps simulation. Thus P44 may be a stable folded protein in aqueous solution. These simulations revealed the presence of 2 local hydrogen bond networks involving intrahelical 3‐center hydrogen bonds in the hydrophobic interior of the coiled coils of GCN4 and P44. The NH hydrogen at d makes a 3‐center hydrogen bond whose major component is to the i — 4 C=O oxygen at g and minor component is to the solvent‐inaccessible i — 3 C=O oxygen at a. Likewise, the NH hydrogen at g makes a 3‐center hydrogen bond with the i — 4 C=O oxygen at c and the buried i — 3 C=O oxygen at d.
Conserved sequence pattern in a wide variety of phosphoesterases
Authors: Koonin Eugene V.
Published: July 2010

A unique sequence pattern, designated the GD/GNH signature, was shown to be conserved in a wide variety of phosphoesterases. The enzymes containing this signature cleave phosphoester bonds in such different substrates as (1) phosphoserine and phosphothreonine in polypeptides; (2) bis(5′‐nucleosidyl)‐tetraphosphates; (3) nucleoside 5′ phosphates; (4) 2′,3′‐cyclic nucleotide phosphates; (5) polynucleotides; (6) 2′‐5′ phospho‐diesters in RNA (intron) lariats; (7) sphingomyelin; and (7) various phosphomonoesters. Two conserved acidic amino acid residues and a conserved histidine residue may be directly involved in phosphoester bond cleavage.
The effect of N‐terminal acetylation on the structure of an N‐terminal tropomyosin peptide and αα‐tropomyosin
Authors: Greenfield Norma J. , Stafford Walter E , Hitchcock‐Degregori Sarah E.
Published: July 2010

We have used a synthetic peptide consisting of the first 30 residues of striated muscle α‐tropomyosin, with GlyCys added to the C‐terminus, to investigate the effect of N‐terminal acetylation on the conformation and stability of the N‐terminal domain of the coiled‐coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low α‐helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N‐terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2‐chained coiled coil at high ionic strength. Disulfide crosslinking and N‐terminal acetylation both greatly stabilized the coiled‐coil α‐helical conformation in high salt.

Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its α‐helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled‐coil formation. In the presence of the organic solvents, N‐terminal acetylation had very little effect on the conformation or stability of the peptide.

Our results indicate that N‐terminal acetylation stabilizes coiled‐coil formation in the peptide. The effect cannot be explained by interactions with the “helix‐dipole” because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N‐terminal acetylation has only small effects on the overall stability of tropomyosin.
Mutational and crystallographic analyses of the active site residues of the bacillus circulans xylanase
Authors: Wakarchuk Warren W. , Campbell Robert L. , Sung Wing L. , Davoodi Jamshid , Yaguchi Makoto
Published: July 2010

Using site‐directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme‐substrate complex to 1.8 Å resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid‐base catalyst in the reaction.
MIF proteins are not glutathione transferase homologs
Authors: Pearson William R.
Published: July 2010

Although macrophage migration inhibitory factor (MIF) proteins conjugate glutathione, sequence analysis does not support their homology to other glutathione transferases. Glutathione transferases are not detected with MIF proteins in searches of protein sequence databases, and MIF proteins do not share significant sequence similarity with glutathione transferases. Homology cannot be demonstrated by multiple sequence alignment or evolutionary tree construction; such methods assume that the proteins being analyzed are homologous.
A proposal for a coherent mammalian histone H1 nomenclature correlated with amino acid sequences
Authors: Parseghian Missag H. , Henschen Agnes H. , Krieglstein Kerstin G. , Hamkalo Barbara A.
Published: July 2010

Bio‐Rex 70 chromatography was combined with reverse‐phase (RP) HPLC to fractionate histone H1° and 4 histone H1 subtypes from human placental nuclei as previously described (Parseghian MH et al., 1993, Chromosome Res 1:127‐139). After proteolytic digestion of the subtypes with Staphylococcus aureus V8 protease, peptides were fractionated by RP‐HPLC and partially sequenced by Edman degradation in order to correlate them with human spleen subtypes (Ohe Y, Hayashi H, Iwai K, 1986, J Biochem (Tokyo) 100:359‐368; 1989, J Biochem (Tokyo) 106:844‐857). Based on comparisons with the sequence data available from other mammalian species, subtypes were grouped. These groupings were used to construct a coherent nomenclature for mammalian somatic Hls. Homologous subtypes possess characteristic patterns of growth‐related and cAMP‐dependent phosphorylation sites. The groupings defined by amino acid sequence also were used to correlate the elution profiles and electrophoretic mobilities of subtypes derived from different species. Previous attempts at establishing an H1 nomenclature by chromatographic or electrophoretic fractionations has resulted in several misidentifications. We present here, for the first time, a nomenclature for somatic Hls based on amino acid sequences that are analogous to those for H1° and Hlt. The groupings defined should be useful in correlating the many observations regarding H1 subtypes in the literature.