bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pressure‐dependent ionization of Tyr 9 in glutathione S‐transferase A1‐1: Contribution of the C‐terminal helix to a “soft” active site
Authors: Atkins William M. , Dietze Eric C. , Ibarra Catherine 
Published: July 2010

AbstractThe glutathione S‐transferase (GST) isozyme A1‐1 contains at its active site a catalytic tyrosine, Tyr 9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS−. In the substrate‐free GST A1‐1, the Tyr 9 has an unusually low pKa, ∼8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan‐free mutant, W21F. In addition, a short α‐helix, residues 208–222, provides part of the GSH and hydrophobic ligand binding sites, and the helix becomes “disordered” in the absence of ligands. Here, hydrostatic pressure has been used to probe the conformational dynamics of the C‐terminal helix, which are apparently linked to Tyr 9 ionization. The extent of ionization of Tyr 9 at pH 7.6 is increased dramatically at low pressures (p1/2 = 0.52 kbar), based on fluorescence titration of Tyr 9. The mutant protein W21F:Y9F exhibits no changes in tyrosine fluorescence up to 1.2 kbar; pressure specifically ionizes Tyr 9. The volume change, ΔV, for the pressure‐dependent ionization of Tyr 9 at pH 7.6, 19°C, was −33 ± 3 mL/mol. In contrast, N‐acetyl tyrosine exhibits a ΔV for deprotonation of −11 ± 1 mL/mol, beginning from the same extent of initial ionization, pH 9.5. The pressure‐dependent ionization is completely reversible for both Tyr 9 and N‐acetyl tyrosine. Addition of S‐methyl GSH converted the “soft” active site to a noncompressible site that exhibited negligible pressure‐dependent ionization of Tyr 9 below 0.8 kbar. In addition, Phe 220 forms part of an “aromatic cluster” with Tyr 9 and Phe 10, and interactions among these residues were hypothesized to control the order of the C‐terminal helix. The amino acid substitutions F220Y, F220I, and F220L afford proteins that undergo pressure‐dependent ionization of Tyr 9 with ΔV values of 31 ± 2 mL/mol, 43 ± 3 mL/mol, and 29 ± 2 mL/mol, respectively. The p1/2 values for Tyr 9 ionization were 0.61 kbar, 0.41 kbar, and 0.46 kbar for F220Y, F220I, and F220L, respectively. Together, the results suggest that the C‐terminal helix is conformationally heterogeneous in the absence of ligands. The conformations differ little in free energy, but they are significantly different in volume, and mutations at Phe 220 control the conformational distribution.
Structural features of the combining site region of Erythrina corallodendron lectin: Role of tryptophan 135
Authors: Adar Rivka , Streicher Hansjörg , Sharon Nathan , Karlsson Karl‐Anders , Ångström Jonas , Moreno Ernesto 
Published: July 2010

AbstractThe role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site‐directed mutagenesis, hemagglutination‐inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl α‐N‐dansylgalactosaminide (MeαGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL‐MeαGalNDns complex. Docking of MeαGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MeαGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MeαGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis‐trans isomerization of the Ala 88‐Asp 89 peptide bond, observed in high‐temperature dynamics simulations, appears not to be a required step.
Modular organization of inteins and C‐terminal autocatalytic domains
Authors: Pietrokovski Shmuel 
Published: July 2010

AbstractAnalysis of the conserved sequence features of inteins (protein “introns”) reveals that they are composed of three distinct modular domains. The N‐terminal (N) and C‐terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C‐terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N‐termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI‐DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing‐endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a “pseudo intein.” The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.
Structural analysis of the multienzyme aminoacyl‐tRNA synthetase complex: A three‐domain model based on reversible chemical crosslinking
Authors: Norcum Mona T. , Warrington J. Anthony 
Published: July 2010

AbstractA subset of eukaryotic aminoacyl‐tRNA synthetases (a‐RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two‐dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K‐RS:p38, D‐RS:K‐RS, R‐RS dimer, K‐RS dimer, K‐RS:Q‐RS, E/P‐RS:K‐RS, E/P‐RS:I‐RS, and Q‐RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D‐RS dimer, R‐RS:p43, D‐RS:Q‐RS, D‐RS:M‐RS, K‐RS:L‐RS, I‐RS:R‐RS, D‐RS:E/P‐RS, I‐RS:Q‐RS, I‐RS:L‐RS. One trimeric association is seen: E/P‐RS:I‐RS:L‐RS.The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl‐tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U‐shaped particle in which each “arm” is considered a domain and the third forms the “base” of the structure. The arms have been termed domain I (D‐RS, M‐RS, Q‐RS) and domain II (K‐RS, R‐RS), with domain III (E/P‐RS, I‐RS, L‐RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three‐dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl‐tRNA synthetase complex.
Subdomain interactions as a determinant in the folding and stability of T4 lysozyme
Authors: Llinás Manuel , Marqusee Susan 
Published: July 2010

AbstractThe folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single‐domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two‐state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N‐terminal subdomain (residues 13‐75), and a C‐terminal subdomain (residues 76‐164 and 1‐12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild‐type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C‐terminal subdomain folds into a marginally stable helical structure, whereas the N‐terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C‐terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full‐length variants. We propose that this acid state is comprised of an unfolded N‐terminal subdomain and a loosely folded C‐terminal subdomain.
Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone
Authors: Bhattacharjya Surajit , Balaram Padmanabhan , Awasthi Satish K. , Adiga P. Radhakantha 
Published: July 2010

AbstractRiboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4‐23 (N21), 170‐186 (R18), and 200‐219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide‐based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure‐stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CαH resonances and the presence of many sequential NH/NH NOEs with many medium‐range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.
Mapping fatty acid binding to β‐lactoglobulin: Ligand binding is restricted by modification of Cys 121
Authors: Narayan Mahesh , Berliner Lawrence J. 
Published: July 2010

AbstractNative β‐lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 μM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4′‐bis 1‐(phenylamino)‐8‐naphthalenesulfonate (bis‐ANS) binding to the protein, indicating that the incorporated appendages do not perturb the internal hydrophobic site within the β‐barrel of Blg (i.e., the retinoid site is unaffected). On the other hand, methylation of Cys 121, reduces the affinity of Blg for palmitic acid by 10‐fold as monitored by intrinsic fluorescence. Modification of the Cys 121 with methyl‐methanethiosulfonate or a thiol‐specific spin label appears to either further weaken or totally eliminate fatty acid binding, respectively, due to steric hindrance. Furthermore, this binding pattern has been independently verified using a spin labeled fatty acid analog and monitoring ESR as well as by bis‐ANS fluorescence when bound to the protein. These results suggest that fatty acids bind at the “external site” of β‐lactoglobulin, between the sole a‐helix and the β‐barrel. In addition, structural stability studies of native and chemically modified Blg appear to confirm this observation as well.
Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon
Authors: Rosenthal R. Scott , Rodwell Victor W. 
Published: July 2010

AbstractThe mvaAB operon of Pseudomonas mevalonii encodes HMG‐CoA reductase (EC 1.1.1.88) and HMG‐CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis‐regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA‐binding activity, comigrated with P15 and P16 during DNA‐affinity chromatography, size‐exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31‐kDa polypeptide complex that contained N‐terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size‐exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N‐termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.
Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy
Authors: Vohník Stanislav , Tuma Roman , Thomas George J. , Hanson Chad , Fuchs James A. , Woodward Clare 
Published: July 2010

AbstractThe active‐site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol‐thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5 ± 0.2, close to values measured previously for wild‐type thioredoxin. Sulfhydryl Raman markers of D26A and wild‐type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side‐chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild‐type protein. Additionally, the thioredoxin main‐chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active‐site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.
How many membrane proteins are there?
Authors: Boyd Dana , Schierle Clark , Beckwith Jon 
Published: July 2010

AbstractOne of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing. In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane. Traditional methods of distinguishing between these two locations have relied on extensive, time‐consuming biochemical studies. The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids. This numerical, sequence‐based approach is usually seen as a first approximation pending more reliable biochemical data. The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved. We have optimized a statistical approach to protein location determination. Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method. We also examine the biological implications of the success of this technique.
Computation of electrostatic complements to proteins: A case of charge stabilized binding
Authors: Chong Lillian T. , Dempster Sara E. , Hendsch Zachary S. , Lee Lee‐Peng , Tidor Bruce 
Published: July 2010

AbstractRecent evidence suggests that the net effect of electrostatics is generally to destabilize protein binding due to large desolvation penalties. A novel method for computing ligand‐charge distributions that optimize the tradeoff between ligand desolvation penalty and favorable interactions with a binding site has been applied to a model for barnase. The result is a ligand‐charge distribution with a favorable electrostatic contribution to binding due, in part, to ligand point charges whose direct interaction with the binding site is unfavorable, but which make strong intra‐molecular interactions that are uncloaked on binding and thus act to lessen the ligand desolvation penalty.
Alignment algorithm for homology modeling and threading
Authors: Alexandrov Nickolai N. , Luethy Roland 
Published: July 2010

AbstractA DNA/protein sequence comparison is a popular computational tool for molecular biologists. Finding a good alignment implies an evolutionary and/or functional relationship between proteins or genomic loci. Sequential similarity between two proteins indicates their structural resemblance, providing a practical approach for structural modeling, when structure of one of these proteins is known. The first step in the homology modeling is a construction of an accurate sequence alignment. The commonly used alignment algorithms do not provide an adequate treatment of the structurally mismatched residues in locally dissimilar regions. We propose a simple modification of the existing alignment algorithm which treats these regions properly and demonstrate how this modification improves sequence alignments in real proteins.
Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling
Authors: Piironen Timo , Villoutreix Bruno O. , Becker Charlotte , Lilja Hans , Hollingsworth Kelly , Vihinen Mauno , Bridon Dominique , Qiu Xiaoxing , Rapp John , Dowell Barry , Lövgren Timo , Pettersson Kim , Lilja Hans 
Published: July 2010

AbstractProstate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237‐amino acid monomeric proteins, with 79% identity in primary structure. Twenty‐five anti‐PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence.Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I‐IV). Binding region I was localized to amino acid residues 1‐13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53‐64, III (a and b) was localized to residues 80‐91 (= kallikrein loop), and IV was localized to residues 151‐164. Mabs binding to regions I and IIa were reactive with free PSA, PSA‐ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA‐ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA‐ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.
Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry
Authors: Wang Fang , Scapin Giovanna , Blanchard John S. , Angeletti Ruth Hogue 
Published: July 2010

AbstractC. glutamicum meso‐diaminopimelate dehydrogenase is an enzyme of the L‐lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme‐substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the “open,” catalytically inactive, conformation but nonexchangeable in the “closed,” catalytically active conformation formed after NADPH and diaminopimelate binding and domain osure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein‐protein interface regions using hydrogen/deuterium exchange and electrospray ionization.