bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of position a in determining the stability and oligomerization state of α‐helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins
Authors: Wagschal Kurt , Tripet Brian , Mant Colin , Hodges Robert S. , Lavigne Pierre
Published: February 2009

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled‐coil protein in determining coiled‐coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three‐stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys‐Gly‐Gly linker, unfolding occurred from the same two‐stranded monomeric oligomerization state for all of the analogs. Denaturation from a two‐stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled‐coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed‐phase high‐performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled‐coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high‐performance size‐exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 μM under benign buffer conditions. The Leu‐, Tyr‐, Gln‐, and His‐substituted analogs were found to be exclusively three‐stranded trimers, while the Asn‐, Lys‐, Orn‐, Arg‐, and Trp‐substituted analogs formed exclusively two‐stranded monomers. Modeling results for the Leu‐substituted analog showed that a three‐stranded oligomerization state is preferred due to increased side‐chain burial, while a two‐stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three‐stranded state.
The complexed structure and antimicrobial activity of a non‐β‐lactam inhibitor of AmpC β‐lactamase
Authors: Powers Rachel A. , Blázquez Jesús , Weston G. Scott , Shoichet Brian K. , Morosini María‐Isabel , Baquero Fernando
Published: February 2009

β‐Lactamases are the major resistance mechanism to β‐lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to β‐lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non‐β‐lactam inhibitors of β‐lactamases were investigated for complementarity to the structure of AmpC β‐lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo (b)thiophene‐2‐boronic acid (BZBTH2B), which inhibited AmpC with a Kit of 27 nM. This inhibitor is chemically dissimilar to β‐lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X‐ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 Å resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1‐amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non‐β‐lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third‐generation cephalosporin against AmpC‐producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against β‐lactams in conjunction with β‐lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many β‐lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure‐based elaboration of this class of non‐β‐lactam inhibitors.
Mutations of endo‐β‐N‐acetylglucosaminidase H active site residues Asp 130 and Glu 132: Activities and conformations
Authors: Rao Vibha , Roey Patrick Van , Cui Tao , Guan Chudi
Published: February 2009

Endo‐β‐N‐acetylglucosaminidase H hydrolyzes the β‐(1‐4)‐glycosidic link of the N,N′‐diacetylchitobiose core of high‐mannose and hybrid asparagine‐linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild‐type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the β‐barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp 130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the β‐barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate‐binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.
Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability‐increasing protein share a similar tertiary structure
Authors: Guyard‐Dangremont Valérie , Tenekjian Viken , Chauhan Vinita , Walter Stephanie , Milne Ross , Roy Patrice , Rassart Eric
Published: February 2009

Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability‐increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand‐binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti‐CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215‐219, 219‐223, 223‐227, and 444‐450, respectively, are predicted to be situated on the external surface of the central β‐sheet and a fifth epitope (residues 225‐258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317‐331, 360‐366, and 393‐410, would form part of the putative carboxyterminal β‐barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.
X‐ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA
Authors: Kurinov I. V. , Rajamohan F. , Venkatachalam T. K. , Uckun F. M.
Published: February 2009

Pokeweed antiviral protein (PAP) is a ribosome‐inactivating protein (RIP), which enzymatically removes a single adenine base from a conserved, surface exposed loop sequence of ribosomal rRNA. We now present unprecedented experimental evidence that PAP can release not only adenine but guanine as well from Escherichia coli rRNA, albeit at a rate 20 times slower than for adenine. We also report X‐ray structure analysis and supporting modeling studies for the interactions of PAP with guanine. Our modeling studies indicated that PAP can accommodate a guanine base in the active site pocket without large conformational changes. This prediction was experimentally confirmed, since a guanine base was visible in the active site pocket of the crystal structure of the PAP‐guanine complex.
A test case for structure‐based functional assignment: The 1.2 Å crystal structure of the yjgF gene product from Escherichia coli
Authors: Volz Karl
Published: February 2009

The YER057c/YIL051c/YjgF protein family is a set of 24 full‐length homologs, each ∼130 residues in length, and each with no known function or relationship to proteins of known structure. To determine the function of this family, the structure of one member–the YjgF protein from Escherichia coli–was solved and refined at a resolution of 1.2 Å. The YjgF molecule is a homotrimer with exact threefold symmetry. Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different. The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related. The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.
Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N‐bromoacetylethanolamine phosphate
Authors: Meng Menghsiao , Chane Tze‐Lieh , Sun Yuh‐Ju , Hsiao Chwan‐Deng
Published: February 2009

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D‐glucopyranose‐6‐phosphate and D‐fructofuranose‐6‐phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N‐bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in kcat/KM, by a factor of 103, indicating the importance of this residue. N‐bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild‐type phosphoglucose isomerase; however, His311 → Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site‐directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of kcat/KM supports a role of His311 as a general base in the catalysis.
Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila
Authors: Martin Stephen R. , Kleinjung Jens , Bayley Peter M. , Lu Alan Q. , Xiao Jie , Beckingham Kathy
Published: February 2009

Androcam is a testis‐specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF‐hand calcium‐binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calciumfree androcam shows a biphasic process characteristic of a two‐domain protein, with the apo‐N‐domain less stable than the apo‐C‐domain. The two EF hands of the C‐domain of androcam bind calcium cooperatively with 40‐fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) ˜ 3 × 103 M−1]. Weak calcium binding is also detected at one or more N‐domain sites. Compared to apo‐calmodulin, apo‐androcam has a smaller conformational response to calcium and a lower α‐helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N‐domain of apo‐androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium‐dependent conformational change in this domain. The affinity of model target peptides for androcam is 103‐105 times lower than for calmodulin, and interaction of the N‐domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium‐induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium‐binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium‐binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N‐domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis‐specific calcium signaling functions.
Real‐time measurements of dark substrate catalysis
Authors: Xie Dong , Suvorov Leonid , Erickson John W. , Gulnik Sergei
Published: February 2009

We have developed a novel procedure to monitor the real‐time cleavage of natural unmodified peptides (dark substrates). In the competition‐based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady‐state enzyme kinetics for a two‐substrate system, we were able to determine both Km and kcat values for cleavage of the dark substrate. The method was applied to HIV‐1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.
Purification and characterization of a cobalt‐activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus
Authors: Cheng Timothy C. , Ramakrishnan Vijay , Chan Sunney I.
Published: February 2009

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C‐terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (Kd = 24 ± 4 μM at 80 °C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and 58 kDa by SDS‐PAGE analysis. In solution, PfuCP exists as a homodimer of ˜ 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90°C under ambient pressure, and a narrow pH optimum of 6.2‐6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far‐UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 °C, its far‐UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far‐UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second‐order decay over time, with 50% activity remaining after ˜ 40 min at 80 °C. The N‐blocked synthetic dipeptide, N‐carbobenzoxy‐Ala‐Arg (ZAR), was used in the purification assay. The kinetic parameters at 80°C with 0.4 mM CoCl2 were: Km, 0.9 ± 0.1 mM; Vmax 2,300 ± 70 U mg−1; and turn over number, 600 ± 20 s−1. Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C‐terminal residues. This broad specificity was confirmed by the C‐terminal ladder sequencing of several synthetic and natural peptides, including porcine N‐acetyl‐renin substrate, for which we have observed (by MALDI‐TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C‐terminus: Ac‐Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐z Phe‐His‐Leu‐Leu‐Val‐Tyr‐Ser.
Locating and identifying posttranslational modifications by in‐source decay during MALDI‐TOF mass spectrometry
Authors: Lennon John J. , Walsh Kenneth A.
Published: February 2009

A technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of cn ion signals generated by in‐source decay during delayed ion extraction in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N, N, N‐trimethyllysine in yeast cytochrome c. A semi‐automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass. A graphical display facilitates illustration of both the location and mass of the PTM.
NikR is a ribbon‐helix‐helix DNA‐binding protein
Authors: Chivers Peter T. , Sauer Robert T.
Published: February 2009

Escherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon‐helix‐helix (β‐α‐α) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N‐terminal domain of NikR show that it has many features expected of a β‐α‐α protein including α‐helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence‐specific manner. Mutation of a residue predicted to be important for DNA‐binding reduces operator affinity but does not affect the secondary structure or stability of the protein.
The relationship between effector binding and inhibition of activity in D‐3‐phosphoglycerate dehydrogenase
Authors: Grant Gregory A. , Xu Xiao Lan , Hu Zhiqin
Published: February 2009

The binding of L‐serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, ˜ 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of ˜ 50% inhibition when only one serine is bound and ˜ 80‐90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 Å apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.
Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer
Authors: Gustavsson Niklas , Härndahl Ulrika , Sundby Cecilia , Emanuelsson Anna , Roepstorff Peter
Published: February 2009

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and α‐crystallins whose members form large oligomeric complexes. The chloroplast‐localized sHsp, Hsp21, contains a conserved methionine‐rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.
2′Halo‐ATP and ‐GTP analogues: Rational phasing tools for protein crystallography
Authors: Gruen Mathias , Becker Christian , Beste Andrea , Reinstein Jochen , Scheidig Axel J. , Goody Roger S.
Published: February 2009

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2′‐halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2′‐substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2′‐halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2′‐halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras (Y32W) (fluorescent mutant) and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of ˜100 (p21ras) or ˜105 (Rab5), and the resulting equilibrium dissociation constants are in the nm or μM range, respectively. The data demonstrate that 2′halo‐ATP and ‐GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 105‐fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the μM range, so that they are still well suited for crystallization of the G‐protein:nucleotide complex.
Common protein architecture and binding sites in proteases utilizing a Ser/Lys dyad mechanism
Authors: Paetzel Mark , J. Strynadka Natalie C.
Published: February 2009

Escherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine‐lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD′ structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage‐site orientation and substrate binding subsites in the UmuD(D′) protease. The structural alignment of SPase and UmuD′ predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases.
Solution structure and dynamics of bovine β‐lactoglobulin A
Authors: Kuwata Kazuo , Era Seiichi , Hoshino Masaru , Forge Vincent , Goto Yuji , Batt Carl A.
Published: February 2009

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine β‐lactoglobulin A at pH 2.0 and 45 °C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight‐stranded continuous antiparallel β‐barrel and one major α‐helix, is similar to the X‐ray dimeric structure obtained at pH 6.2, including βIstrand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. {1H}‐15N NOE revealed that βf, βg, and βH strands buried under the major α‐helix are rigid on a pico‐ to nanosecond time scale and also emphasized rapid fluctuations of loops and the N‐ and C‐terminal regions.
The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: Implications for drug design
Authors: Mahadevan Daruka , Saldanha José W.
Published: February 2009

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS‐16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate‐specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three‐dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.
Acetylcholinesterase of schistosoma mansoni—Functional correlates: Contributed in honor of Professor Hans Neurath's 90th birthday
Authors: Arnon Ruth , Tarrab‐Hazdai Rebeca , Silman Israel
Published: February 2009

Acetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of ∼6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are “true” acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin‐binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface‐localized enzyme on the intact organism, and produce almost total complement‐dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.
NMR assignments, secondary structure, and global fold of calerythrin, an EF‐hand calcium‐binding protein fromSaccharopolyspora erythraea
Authors: Aitio Helena , Heikkinen Sami , Äinen Ilkka Kilpel , Annila Arto , Thulin Eva , Drakenberg Torbjörn
Published: February 2009

Calerythrin is a 20 kDa calcium‐binding protein isolated from gram‐positive bacteriumSaccharopolyspora erythraea.Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF‐hand calcium‐binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain1H,13C, and15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]‐edited TOCSY, and HCCH‐TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N‐H residual dipolar couplings were measured from a spin‐state selective [1H,15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF‐hand motifs with characteristic helix‐loop‐helix patterns were observed. Three of these are typical calcium‐binding EF‐hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium‐binding proteins from the sandwormNereis diversicolorand the amphioxusBranchiostoma lanceolatum.
Rotamer strain as a determinant of protein structural specificity
Authors: Lazar Greg A. , Handel Tracy M. , Johnson Eric C. , Desjarlais John R.
Published: February 2009

We present direct evidence for a change in protein structural specificity due to hydrophobic core packing. High resolution structural analysis of a designed core variant of ubiquitin reveals that the protein is in slow exchange between two conformations. Examination of side‐chain rotamers indicates that this dynamic response and the lower stability of the protein are coupled to greater strain and mobility in the core. The results suggest that manipulating the level of side‐chain strain may be one way of fine tuning the stability and specificity of proteins.
Structural determinants of trypsin affinity and specificity for cationic inhibitors
Authors: Polticelli Fabio , Honig Barry , Ascenzi Paolo , Bolognesi Martino
Published: February 2009

The binding free energies of four inhibitors to bovine β‐trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson‐Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy‐surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine‐ and lysine‐based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp 189 in the inhibitor‐free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor‐free state, thus reducing the entropie cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.
Structure of the fibrinogen γ‐chain integrin binding and factor XIIIa cross‐linking sites obtained through carrier protein driven crystallization
Authors: Ware Scott , Anderson Wayne F. , Donahue John P. , Hawiger Jacek
Published: February 2009

The human fibrinogen γ‐chain C‐terminal segment functions as the platelet integrin binding site as well as the Factor XIIIa cross‐linking substrate and thus plays an important role in blood clot formation and stabilization. The three‐dimensional structure of this segment has been determined using carrier protein driven crystallization. The C‐terminal segment, γ‐(398–411), was attached to a linker sequence at the C‐terminus of glutathione S‐transferase and the structure of this fusion protein determined at 1.8 Å resolution. Functional studies of the chimeric protein demonstrate that the fibrinogen sequence in the presence of the carrier protein retains its specific functions as ligand for platelet integrin αIIbβ3 (gpIIb/IIIa) and as a cross‐linking substrate for Factor XIIIa. The structure obtained for the fibrinogen γ‐chain segment is not affected by crystal packing and can provide the missing links to the recently reported model of cross‐linked fibrin.
Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus
Authors: Sakowicz Roman , Farlow Sam , Goldstein Lawrence S.B.
Published: February 2009

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIFl, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus‐directed microtubule movement up to 2 μm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.
Helix‐bundle membrane protein fold templates
Authors: Bowie James U.
Published: February 2009

In the fold recognition approach to structure prediction, a sequence is tested for compatibility with an already known fold. For membrane proteins, however, few folds have been determined experimentally. Here the feasibility of computing the vast majority of likely membrane protein folds is tested. The results indicate that conformation space can be effectively sampled for small numbers of helices. The vast majority of potential monomeric membrane protein structures can be represented by about 30‐folds for three helices, but increases exponentially to about 1, 500, 000 folds for seven helices. The generated folds could serve as templates for fold recognition or as starting points for conformational searches that are well distributed throughout conformation space.
Protein structure determination using a database of interatomic distance probabilities
Authors: Wall Michael E. , Phillips George N. , Subramaniam Shankar
Published: February 2009

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X‐ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side‐chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side‐chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high‐resolution refinements were improved, as is shown by an R‐factor analysis. The improvements were greatest when refinements were carried out using X‐ray data truncated at 3.5 Å. The database potential should therefore be a valuable tool for determining X‐ray structures, especially when only low‐resolution data are available.
Influence of key residues on the reaction mechanism of the cAMP‐dependent protein kinase
Authors: Hutter Michael C. , Helms Volkhard
Published: February 2009

The reaction mechanism of the catalytic phosphoryl transfer of cAMP‐dependent protein kinase (cAPK) was investigated by semi‐empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol−1 and the reaction itself to be exothermic by 12.2 kcal mol−1. The active site residue Asp 166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two‐dimensional energy hypersurfaces of the protein kinase wild‐type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys 168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.
Comparison of the kinetics of S‐S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme
Authors: Roux Pascale , Chaffotte Alain‐François , Goldberg Michel E. , Ruoppolo Margherita
Published: February 2009

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray‐mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far‐UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.
Effect of hydrostatic pressure on unfolding of α‐lactalbumin: Volumetric equivalence of the molten globule and unfolded state
Authors: Kobashigawa Yoshihiro , Nitta Katsutoshi , Sakurai Masao
Published: February 2009

The effect of pressure on the unfolding of bovine α‐lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, ΔV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The ΔV was estimated to be −63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that ΔV was −66 cm3/mol at 25 °C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of α‐lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.
Mass spectral characterization of a protein—nucleic acid photocrosslink
Authors: Golden Mace C. , Resing Katheryn A. , Koch Tad H. , Collins Brian D. , Willis Michael C.
Published: February 2009

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61‐mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross‐linked protein‐nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130–138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.
Pressure‐induced thermostabilization of glutamate dehydrogenase from the hyperthermophile pyrococcus furiosus
Authors: Sun MICHAEL M.C. , Tolliday Nicola , Vetriani Costantino , Robb Frank T. , Clark Douglas S.
Published: February 2009

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophilePyrococcus furiosus(Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C‐terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin‐label attached to the terminal cysteine of rGDHtrevealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion‐pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36‐fold for rGDHtat 105 C and 750 atm, the largest pressure‐induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHtwas also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure‐induced thermostabilization is proposed.
The calorimetric criterion for a two‐state process revisited
Authors: Zhou Yaoqi , Hall Carol K. , Karplus Martin
Published: February 2009

The “calorimetric criterion” is one of the important experimental approaches for determining whether protein folding is an “all‐or‐none” two‐state transition (i.e., whether intermediates are present at equilibrium). The calorimetric criterion states that the equivalence of the “measured” calorimetric enthalpy change and the effective two‐state van't Hoff enthalpy change demonstrates that there is a two‐state transition. This paper addresses the essential question of whether the calorimetric criterion is a necessary and sufficient condition for a two‐state process and shows that it is necessary but not sufficient by means of specific examples. Analysis of simple models indicates that the heat capacity curve, regardless of whether it originates from a two‐state process or not, can always be decomposed in such a way that the calorimetric criterion is satisfied. Exact results for a three‐state model and a homopolymer tetramer demonstrate that the deviation from the calorimetric criterion is not simply related to the population of intermediate states. Analysis of a three‐helix bundle protein model, which has a two‐state folding from a random coil to ordered (molten) globule, shows that the calorimetric criterion may not be satisfied if the standard linear interpolation of baselines (weighted or unweighted) is employed. A specific example also suggests that the more recently introduced deconvolution method is not necessarily better than the simple calorimetric criterion for distinguishing a two‐state transition from a three‐state transition. Although the calorimetric criterion is not a sufficient condition for a two‐state process, it is likely to continue to be of practical utility, particularly when its results are shown to be consistent with those from other experimental methods.