bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies
Authors: Speed Margaret A. , Morshead Timothy , Wang Daniel I.C. , King Jonathan
Published: July 2010

The partitioning of partially folded polypeptide chains between correctly folded native states and off‐pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (Speed MA, Wang DIC, King J. 1995. Protein Sci 4:900–908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi‐Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945–15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off‐pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N‐terminus, indicating that the N‐terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N‐terminal epitope did not, indicating that the conformation of the N‐terminus is not a key determinant of the productive folding and chain association pathway.
Crystal structure of the reduced Schiff‐base intermediate complex of transaldolase B from escherichia coli: Mechanistic implications for class I aldolases
Authors: Jia Jia , Lindqvist Ylva , Schneider Gunter , Schörken Ulrich , Sprenger Georg A.
Published: July 2010

Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff‐base intermediate between dihydroxyacetone and the ϵ‐amino group of a lysine residue at the active site of the enzyme is formed. This Schiff‐base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 Å resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.
Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue
Authors: Friedland Diana E. , Goss Dixie J. , Shoemaker Michael T. , Xie Yiping , Wang Yuhuan , Hagedorn Curt H.
Published: July 2010

Binding of eIF‐4E to the 5′ m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the moiecular basis for this recognition, photoaffinity labeling with [γ‐32P]8‐N3GTP was used in binding site studies of human recombinant cap binding protein reIF‐4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [γ‐32P]8‐N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)‐chelate chromatography and reverse‐phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled reIF‐4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113‐Arg 122. Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [γ‐32P]8‐N3GTP. A detailed understanding of eIF‐4E‐m7G mRNA cap interactions may lead the way to regulating this essential protein‐RNA interaction for specific mRNA in vivo.
3‐Hydroxy‐3‐methylglutaryl‐coenzyme A reductase of haloferax volcanii: Role of histidine 398 and attenuation of activity by introduction of negative charge at position 404
Authors: Bischoff Kenneth M. , Rodwell Victor W.
Published: July 2010

Mutant 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG‐CoA reductases via charge‐charge interaction with the active site histidine. To first verify the sequence‐based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG‐CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG‐CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403‐residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii‐hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild‐type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.
γ‐Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function
Authors: Gillis Shmuel , Furie Barbara C. , Furie Bruce , Patel Himakshi , Huberty Michael C. , Switzer Mary , Barry Foster W. , Scoble Hubert A. , Bond Michael D.
Published: July 2010

The γ‐carboxyglutamic acid (Gla) domains of the vitamin K‐dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion‐exchange chromatography, peptide mapping, mass spectrometry, and N‐terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/γ40E is uncarboxylated at residue 40 and factor IX/γ36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully y‐carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma‐derived factor IX. As monitored by anti‐factor IX:Ca(II)‐specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)‐induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half‐maximal inhibition of the binding of 125I‐labeled plasma‐derived factor IX at concentrations of 2–6 nM. Functionally, factor IX/γ36,40E and factor IX/γ40E were similar to fully γ‐carboxylated recombinant factor IX and plasma‐derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti‐factor IX:Mg(II)‐specific antibodies because these antibodies bound factor IX preferentially to factor LX/γ36,40E and factor IX/γ40E. These results demonstrate that the γ‐carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.
Mechanism of the stabilization of ribonuclease a by sorbitol: Preferential hydration is greater for the denatured than for the native protein
Authors: Xie Guifu , Timasheff Serge N.
Published: July 2010

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, δΔG° = ΔG°(sorbitol) ‐ ΔG°(water). Measurements of preferential interactions at 48 °C at pH 5.5, where protein is native, and pH 2.0, where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, ΔμN

2 > Δμ
N

2, which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co‐solvent on denaturation, ΔG°

w + Δμ
D
2 = ΔG°
S + Δμ
D

2 indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water co‐solvent exchange at thermodynamically non‐neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.
Temperature dependence of the preferential interactions of ribonuclease a in aqueous co‐solvent systems: Thermodynamic analysis
Authors: Xie Guifu , Timasheff Serge N.
Published: July 2010

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co‐solvents, more so at low pH than high pH (except 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co‐solvents was high at temperatures below 30 °C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 °C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (∂μ2/∂m3)T, P, m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, \documentclass{article}\pagestyle{empty}\begin{document}$ \Delta \bar H_{2,tr} $\end{document}, and transfer entropy \documentclass{article}\pagestyle{empty}\begin{document}$ \Delta \bar S_{2,tr} $\end{document}. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water to a 30% sorbitol solution.
A new method for analyzing protein sequence relationships based on Sammon maps
Authors: Agrafiotis Dimitris K.
Published: July 2010

Recent advances in gene sequencing and rational drug design have re‐emphasized the need for new methods for protein analysis, classification, and structure and function prediction. In this article, we introduce a new method for analyzing protein sequences based on Sammon's non‐linear mapping algorithm. When applied to a family of homologous sequences, the method is able to capture the essential features of the similarity matrix, and provides a faithful representation of chemical or evolutionary distance in a simple and intuitive way. The merits of the new algorithm are demonstrated using examples from the protein kinase family.
Three‐dimensional structures of three engineered cellulose‐binding domains of cellobiohydrolase I from Trichoderma reesei
Authors: Mattinen Maija‐Liisa , Kontteli Maarit , Kerovuo Janne , Drakenberg Torbjörn , Annila Arto , Linder Markus , Reinikainen Tapani , Lindeberg Gunnar
Published: July 2010

Three‐dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel β‐sheet structure of the CBD fold was preserved in all engineered peptides. The three‐dimensional NMR‐based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N‐terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.
The NMR side‐chain assignments and solution structure of enzyme HBcellobiose of the phosphoenolpyruvate‐dependent phosphotransferase system of Escherichia coli
Authors: Ab Eiso , Schuurman‐Wolters Gea , Dijkstra Klaas , Scheek Ruud M. , Robillard George T. , Reizer Jonathan , Saier Milton H.
Published: July 2010

The assignment of the side‐chain NMR resonances and the determination of the three‐dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate‐dependent phosphotransferase system of Escherichia coli are presented. The side‐chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH‐TOCSY, HCCH‐COSY, and COCCH‐TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments.

In order to obtain the three‐dimensional structure, NOE data were collected from l5N‐NOESY‐HSQC, 13C‐HSQC‐NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 Å on Cα atoms. IIBce consists of a four stranded parallel β‐sheet, in the order 2134. The sheet is flanked with two and three α‐helices on either side. Residue 10, a cysteine in the wild‐type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first β‐strand. A loop that is proposed to be involved in the binding of the phosphoryl‐group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.
Analysis of protein structure in intact cells: Crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor
Authors: Hughson Andrew G. , Hazelbauer Gerald L. , Lee Geoffrey F.
Published: July 2010

Oxidative crosslinking of cysteines introduced by site‐specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)‐(o‐phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining trans‐membrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1′) in the other subunit and a more distant placement of TM2 and TM2′, the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1‐TM1′ interaction and TM2‐TM2′ orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.
A novel methodology for assignment of disulfide bond pairings in proteins
Authors: Wu Jiang , Watson J. Throck
Published: July 2010

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2‐carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by l‐cyano‐4‐dimethylamino‐pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed‐phase HPLC, are subjected to cleavage of the peptide bonds on the N‐terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI‐MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, β‐elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.
Cooperative α‐helix formation of β‐lactoglobulin and melittin induced by hexafluoroisopropanol
Authors: Hirota Nami , Goto Yuji , Mizuno Kazuko
Published: July 2010

Alcohols denature the native state of proteins, and also stabilize the α‐helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine β‐lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, β‐lactoglobulin exhibited a transformation from the native state, consisting of β‐sheets, to the α‐helical state, whereas melittin folded from the unfolded state to the α‐helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol‐induced transitions were analyzed assuming a two‐state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle‐like clusters of HFIP is important.
The role of helix VIII in the lactose permease of Escherichia coli: II. Site‐directed sulfhydryl modification
Authors: Frillingos Stathis , Kaback H. Ronald
Published: July 2010

Cys‐scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S, Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431–437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N‐ethylmaleimide (NEM)‐sensitive mutant is examined in the absence or presence of β,D‐galactopyranosyl l‐thio‐β,D‐galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264 → Cys, Gly 268 → Cys, and Asn 272 → Cys, and alkylation of these single‐Cys mutants in right‐side‐out membrane vesicles with [l4C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265 → Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM‐labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262 → Cys, Glu 269 → Cys, Ala 273 → Cys, Met 276 → Cys, Phe 277 → Cys, or Ala 279 → Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand‐induced conformational change.
Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases
Authors: Zhou Genfa , Parthasarathy Gopalakrishnan , Somasundaram Thayumanasamy , Ables Andrea , Roy Lance , Chapman Michael S. , Strong Scott J. , Ellington W. Ross
Published: July 2010

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated fromE. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 Å resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection atcryotemperatures have all yielded significant improvements in diffraction.
Thermoanaerobacter brockii alcohol dehydrogenase: Characterization of the active site metal and its ligand amino acids
Authors: Bogin Oren , Peretz Moshe , Burstein Yigal
Published: July 2010

The active‐site metal ion and the associated ligand amino acids in the NADP‐linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site‐directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 25:6549–6555) were verified. To determine the role of the putative active‐site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (<5%). Site‐directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non‐chelating residue, alanine, abolished not only the metal‐binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc‐binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.
Evidence for PDZ domains in bacteria, yeast, and plants
Authors: Ponting Christopher P.
Published: July 2010

Several dozen signaling proteins are now known to contain 80‐100 residue repeats, called PDZ (or DHR or GLGF) domains, several of which interact with the C‐terminal tetrapeptide motifs X‐Ser/Thr‐X‐Val‐COO− of ion channels and/or receptors. PDZ domains have previously been noted only in mammals, flies, and worms, suggesting that the primordial PDZ domain arose relatively late in eukaryotic evolution. Here, techniques of sequence analysis—including local alignment, profile, and motif database searches—indicate that PDZ domain homologues are present in yeast, plants, and bacteria. It is suggested that two PDZ domains occur in bacterial high‐temperature requirement A (htrA) and one in tail‐specific protease (tsp) homologues, and that a yeast htrA homologue contains four PDZ domains. Sequence comparisons suggest that the spread of PDZ domains in these diverse organisms may have occurred via horizontal gene transfer. The known affinity of Escherichia coli tsp for C‐terminal polypeptides is proposed to be mediated by its PDZ‐like domain, in a similar manner to the binding of C‐terminal polypeptides by animal PDZ domains.
Remodeling domain interfaces to enhance heterodimer formation
Authors: Zhu Zhenping , Presta Leonard G. , Zapata Gerardo , Carter Paul
Published: July 2010

An anti‐p185HER2/anti‐CD3 humanized bispecific diabody was previously constructed from two cross‐over single‐chain Fv in which VH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide‐stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti‐p185HER2 VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide‐stabilized compared to the parent diabody (>96% versus 72%), whereas the overall yield was >60‐fold lower. Eleven “knob‐into‐hole” diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti‐p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.
The kinetic basis for the stabilization of staphylococcal nuclease by xylose
Authors: Frye Kelly J. , Royer Catherine A.
Published: July 2010

The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence‐detected pressure‐jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234–10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909–4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate‐limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.
Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site
Authors: Weaver Todd , Lees Mason , Banaszak Leonard
Published: July 2010

Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C‐terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A‐ and B‐, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A‐site resulted in a large decrease in specific activity, while the H129N mutation at the B‐site had essentially no effect. From the results, we conclude that the A‐site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B‐site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.
Monitoring calcium‐induced conformational changes in recoverin by electrospray mass spectrometry
Authors: Neubert Thomas A. , Walsh Kenneth A. , Hurley James B. , Johnson Richard S.
Published: July 2010

Recoverin is a calcium‐binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N‐acylated by myristoyl and related fatty acyl residues that are thought to act as “calcium‐myristoyl switches,” whereby, in the presence of Ca2+, the N‐terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium‐binding sites. In addition to supporting the calcium‐myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N‐acyl group (N‐lauroyl or N‐myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X‐ray crystallography.
An electrophile‐nucleophile interaction in metalloprotein structures
Authors: Chakrabarti Pinak , Pal Debnath
Published: July 2010

A new attractive interaction in metalloprotein structures, between the thiolate anion of a metal‐bound cysteine (acting as a nucleophile) and a carbonyl carbon of a peptide group (an electrophile), has been identified. From 82 cases extracted from 23 metalloprotein structures, the interacting S and C atoms are found to be at a distance of 3.2 (±2) Å, such that the angle S···C — O is 109° (±15°). Usually, the interacting atoms are from the same Cys residue, and to allow the S to interact with the carbonyl group the side‐chain and the main‐chain torsion angles deviate from those found in cysteines not bound by metals. There is a good correlation between the S···C distance and the angular deviation of the S···C vector from the normal to the peptide plane. Various data points may be envisaged to represent “snapshots” along the reaction coordinate for the intra‐residue attack of Cys S on the CO group.
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

The 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

The 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

Analysis 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

A 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

The 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

Riboflavin 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.