bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating the energetics of empty cavities and internal mutations in proteins
Authors: Rashin Alexander A. , Rashin Bella H. , Rashin Annita , Abagyan Ruben
Published: February 2009

The energetics of cavity formation in proteins is evaluated with two different approaches and results are analyzed and compared to experimental data. In the first approach, free energy of cavity formation is extracted by RMS fitting from the distribution of numbers of cavities, N, with different volumes, Vcav' in 80 high‐resolution protein structures. It is assumed that the distribution of number of cavities according to their volume follows the Boltzmann law, NVcav = exp[(‐α.Vcav b)/kT], or its simplified form. Specific energy cost of cavity formation, α, extracted by RMS fitting from these distributions is compared to α values extracted from experimental free energies of cavity formation in T4 lysozyme fitted to similar expressions. It is found that fitting of both sets of data leads to similar magnitudes and uncertainties in the calculated free energy values. It is shown that Boltzmann‐like distribution of cavities can be derived for a simple model of an equilibrium interconversion between mutants in an extracellular system. We, however, suggest that a partitioning into cavity‐dependent and cavity‐independent terms may lose meaning when one attempts to describe mutation effects on protein stability in terms of specific free energy contributions. As an alternative approach, a direct molecular mechanics evaluation is attempted of T4 lysozyme destabilization by five single cavity‐creating mutations. The calculations are based on the approach used in calculations of the energetics of packing defects in crystals. For all mutations calculated destabilizations agree with the corresponding experimental values within +0.6 kcal/mol. A computational relaxation of the mutant was most difficult to achieve for the mutation producing the smallest cavity. However, calculations do not always reproduce crystallographically observed contraction/expansion of cavities. It is suggested that this may be related to usually observed large RMS differences (>1 Å) between crystallographic and energy‐minimized protein structures, and thus correct energetics might be easier to calculate than the correct geometry.
Dissection of the gene of the bifunctional PGK‐TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: Design and characterization of the separate triosephosphate isomerase
Authors: Beaucamp Nicola , Hofmann Alexandra , Kellerer Barbara , Jaenicke Rainer
Published: February 2009

Triosephosphate isomerase (TIM), from the hyperthemophilic bacterium Thermotoga maritima, has been shown to be covalently linked to phosphoglycerate kinase (PGK) forming a bifunctional fusion protein with TIM as the C‐terminal portion of the subunits of the tetrameric protein (Schurig et al., EMBO J 14:442‐451, 1995). To study the effect of the anomalous state of association on the structure, stability, and function of Thermotoga TIM, the isolated enzyme was cloned and expressed in Escherichia coli, and compared with its wild‐type structure in the PGK‐TIM fusion protein. After introducing a start codon at the beginning of the tpi open reading frame, the gene was expressed in E.c.BL21(DE3)/ pNBTIM. The nucleotide sequence was confirmed and the protein purified as a functional dimer of 56.5 kDa molecular mass. Spectral analysis, using absorption, fluorescence emission, near‐ and far‐UV circular dichroism spectroscopy were used to compare the separated Thermotoga enzyme with its homologs from mesophiles. The catalytic properties of the enzyme at ∼ 80°C are similar to those of its mesophilic counterparts at their respective physiological temperatures, in accordance with the idea that under in vivo conditions enzymes occupy corresponding states. As taken from chaotropic and thermal denaturation transitions, the separated enzyme exhibits high intrinsic stability, with a half‐concentration of guanidinium‐chloride at 3.8 M, and a denaturation half‐time at 80° C of 2 h. Comparing the properties of the TIM portion of the PGK‐TIM fusion protein with those of the isolated recombinant TIM, it is found that the fusion of the two enzymes not only enhances the intrinsic stability of TIM but also its catalytic efficiency.
Evidence for two conformational states of thioredoxin reductase from Escherichia coli: Use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation
Authors: Mulrooney Scott B. , Williams Charles H.
Published: February 2009

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two‐stranded β sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox‐active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66° relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800‐816), placing either the redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active‐site mutant. The flavin fluorescence of this mutant is only 7% that of wild‐type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active‐site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5‐fold increase in fluorescence. Titration of the PMA‐treated mutant with the nonreducing NADP(H) analogue, 3‐aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild‐type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA‐treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.
Conformationally driven protease‐catalyzed splicing of peptide segments: V8 protease‐mediated synthesis of fragments derived from thermolysin and ribonuclease A
Authors: Kumaran Sangaralingam , Datta Debjani , Roy Rajendra P.
Published: February 2009

We have studied the conformation as well as V8 protease‐mediated synthesis of peptide fragments, namely amino acid residues 295‐316 (TC‐peptide) of thermolysin and residues 1‐20 (S‐peptide) of ribonuclease A, to examine whether “conformational trapping” of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent‐mediated cooperative helix formation in TC‐peptide with attainment of about 30‐35% helicity in the presence of 40% 1‐propanol and 2‐propanol solutions at pH 6 and 4°C. The thermal melting profiles of TC‐peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC‐peptide from a 1:1 mixture of the noninteracting complementary fragments (TC295‐302 and TC303‐316) in the presence of the above cosolvents at pH 6 and 4°C. In contrast, V8 protease did not catalyze the ligation of S1‐9 and S10‐20, although S‐peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S‐peptide (SA‐peptide) in which residues 10‐14 were substituted (RQHMD → VAAAK). While S‐peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA‐peptide displayed predominantly β‐sheet conformation. SA‐peptide showed enhanced resistance to proteolysis as compared with S‐peptide. Thus, failure of semisynthesis of S‐peptide may be a consequence of high flexibility around the 9‐10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease‐mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.
Three‐dimensional solution structure of β cryptogein, a β elicitin secreted by a phytopathogenic fungus phytophthora cryptogea
Authors: Fefeu Sandrine , Bouaziz Serge , Guittet Eric , Huet Jean‐Claude , Pernollet Jean‐Claude
Published: February 2009

Cryptogein belongs to a new family of 10‐kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive‐like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of β cryptogein from Phytophthoru cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE‐derived distance restraints and 40 dihedral angle restraints obtained from 3JHNHα couplings. The RMS deviation from the mean structure is 0.87 ± 0.14 Å for backbone atoms and 1.34 ± 0.14 Å for all the non‐hydrogen atoms of residues 2 to 98. The structure of β cryptogein reveals a novel protein fold, with five helices and a double‐stranded β‐sheet facing an Ω‐loop. One edge of the β‐sheet and the adjacent face of the Ω‐loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X‐ray structure, with slight differences lightly due to the crystal packing.
Anatomy of a conformational transition of β‐strand 6 in soybean P‐amylase caused by substrate (or inhibitor) binding to the catalytical site
Authors: Pujadas Gerard , Palau Jaume
Published: February 2009

A computational study of the five soybean β‐amylase X‐ray structures reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the β‐strand 6 (residues 341‐343) in the (β/α)8 molecular scaffold. Backbone distortions that involve considerable changes in the ø and ψ angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA‐layer (at the C‐terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand‐binding process, there is a negative enthalpy change for these residues (—27 and —34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed “stability‐function” hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452‐456). They are also in good agreement with other experimental results in the literature that describe the role of the 341‐343 segment in β‐amylase activity. Site‐directed mutagenesis focused on these residues could be useful for undertaking functional studies of β‐amylase.
Pilin C‐terminal peptide binds asialo‐GM1 in liposomes: A 2H‐NMR study
Authors: Jones David H. , Barber Kathryn R. , Grant Chris W. M. , Hodges Robert S.
Published: February 2009

Wideline 2H‐NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128‐144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo‐GM1) carried 2H probe nuclei on the terminal and next‐to‐terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 104 s−1. Spectral effects were reversed by reduction of the single peptide disulfide bond—a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:6255‐16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.
Preliminary structural studies on the multi‐ligand‐binding domain of the transcription activator, BmrR, from bacillus subtilis
Authors: Zheleznova Ekaterina E. , Brennan Richard G. , Markham Penelope N. , Neyfakh Alexander A.
Published: February 2009

In the bacterium Bacillus subtilis, the DNA‐binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144‐amino acid‐residue carboxy terminal dimerization/ ligand‐binding domain of the BmrR, named the BRC (BmrR C‐terminus). Tetragonal crystals of ligand‐free BRC take the space group P41212, or its enantiomorph P43212, with unit cell dimensions a = b = 76.3 Å, c = 96.0 Å, α = β = γ = 90°. Diffraction is observed to at least 2.7 Å resolution at room temperature. In addition, we determined the secondary structure content of ligand‐free and rhodamine‐bound BRC by circular dichroism. In the ligand‐free form, BRC has considerable β‐sheet content (41%) and little α‐helix structure (13%). After BRC binds rhodamine, its β‐sheet content increases to 47% while the α‐helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.
Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci
Authors: Devulapalle Kumari S. , Goodman Steven D. , Gao Qian , Hemsley Ann , Mooser Gregory
Published: February 2009

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of α‐amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge‐based interactive algorithm, MACAW, which provided significant level of homology with α‐amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/α‐amylase residues conserved in all but one α‐amylase invariant residues. Site‐directed mutagenesis of the three GTF catalytic residues are homologous with the α‐amylase catalytic triad. The glucosyltransferases are members of the 4/7‐superfamily that have a (β/α)8‐barrel structure and belong to family 13 of the glycohydralases.
Structural characterization of human hemoglobin crosslinked by bis(3,5‐dibromosalicyl) fumarate using mass spectrometric techniques
Authors: Yu Zhonghua , Friso Giulia , Miranda Jj , Burlingame Alma L. , Patel Mehul J. , Lo‐Tseng Thomas , Moore Edwin G.
Published: February 2009

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric‐based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5‐dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size‐exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP‐HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin β‐chain, and two hemoglobin α‐chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.
Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan‐synthase β chain
Authors: Gast Klaus , Mueller‐Frohne Marlies , Chaffotte Alain F. , Guillou Yvonne , Hodges Maria , Cadieux CÉLine , Goldberg Michel E. , Zirwer Dietrich , Damaschun Gregor
Published: February 2009

The isolated, 101‐residue long C‐terminal (so called F2) fragment of the β chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far‐UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60°C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.
α‐Hemolysin, γ‐hemolysin, and leukocidin from Staphylococcus aureus: Distant in sequence but similar in structure
Authors: Gouaux Eric , Hobaugh Michael , Song Langzhou
Published: February 2009

α‐Hemolysin from Staphylococcus aureus assembles from a water‐soluble, monomeric species to a membrane‐bound heptamer on the surface of target cells, creating water‐filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the γ‐hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of α‐hemolysin, γ‐hemolysin, and leukocidin in the context of the α‐hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between α‐hemolysin and the γ‐hemolysin and leukocidin toxins is in the so‐called twilight zone, the three‐dimensional structures of the protomers are probably conserved. By analogy with α‐hemolysin, γ‐hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an anti‐parallel β‐barrel constitutes the primary membrane‐embedded domain.
Crystal structures of the cadmium‐ and mercury‐substituted metallo‐β‐lactamase from Bacteroides fragilis
Authors: Concha Nestor O. , Herzberg Osnat , Rasmussen Beth A. , Bush Karen
Published: February 2009

The metallo‐β‐lactamases require zinc or cadmium for hydrolyzing β‐lactam antibiotics and are inhibited by mercurial compounds. To date, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn2+‐bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the β‐lactam ring. To study the metal coordination further, the crystal structures of a Cd2+‐bound enzyme and of an Hg2+‐soaked zinc‐containing enzyme have been determined at 2.I Å and 2.7 Å, respectively. Given the diffraction resolution, the Cd2+‐bound enzyme exhibits the same active‐site architecture as that of the Zn2+‐bound enzyme, consistent with the fact that both forms are enzymatically active. The 10‐fold reduction in activity of the Cd2+‐bound molecule compared with the Zn2+‐bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg2+‐bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2‐Zn2+‐bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 Å away from Zn1 and 3.9 Å away from the site where Zn2 is located in the 2‐Zn2+‐bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.
Hinge bending within the cytokine receptor superfamily revealed by the 2.4 Å crystal structure of the extracellular domain of rabbit tissue factor
Authors: Muller Yves A. , Kelley Robert F. , De Vos Abraham M.
Published: February 2009

Tissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF‐FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 Å resolution and refined to a crystallographic R‐value of 19.1% (free R‐value, 27.7%). Like the human homologue, the extracellular portion consists of two fibronectin type III domains connected by a short α‐helical segment. Unexpectedly, the two molecules in the crystallographic asymmetric unit differ in their relative domain‐domain orientation, revealing unsuspected hinge motion consisting of a rotation of about 12.7° around an axis intersecting the linker segment at residue 106. Superposition of rabbit tissue factor with free and bound human tissue factor allows for the detection of an identical, albeit smaller, hinge motion in human TF induced upon binding of FVIIa. This raises the possibility that a very similar hinge axis may be present in other members of the cytokine receptor superfamily.
A thioredoxin fusion protein of VanH, a D‐lactate dehydrogenase from Enterococcus faecium: Cloning, expression, purification, kinetic analysis, and crystallization
Authors: Stoll Vincent S. , Manohar A. Vaito , Gillon Wanda , Macfarlane Emma L. A. , Hynes Rosemary C. , Pai Emil F.
Published: February 2009

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D‐lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on kcat/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of ∼ 3.5 μ, 19.0 μ, and 1.5 μ, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408‐10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP‐ribose, NAD +, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash‐frozen crystals of the tetragonal form diffracted to 3.0 Å resolution at a synchrotron radiation source.
Production, crystallization, and preliminary X‐ray analysis of the human MHC class Ib molecule HLA‐E
Authors: O'callaghan Christopher A. , McMichael Andrew J. , Bell John I. , Blundell Charlie D. , Jakobsen Bent K. , Willcox Benjamin E. , Tormo José , Stuart David I. , Jones E. Yvonne
Published: February 2009

HLA‐E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA‐E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA‐E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA‐E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA‐E forms a stable complex with β2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 Å with space group P3I21 and unit cell dimensions a = 182.2 Å, b = 182.2 Å, c = 88.4 Å.
Crystal structure of cis‐biphenyl‐2,3‐dihydrodiol‐2,3‐dehydrogenase from a PCB degrader at 2.0 Å resolution
Authors: Hülsmeyer Martin , Hecht Hans‐Jürgen , Niefind Karsten , Schomburg Dietmar , Hofer Bernd , Timmis Kenneth N. , Eltis Lindsay D.
Published: February 2009

cis‐Biphenyl‐2,3‐dihydrodiol‐2,3‐dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+‐enzyme complex was determined by molecular replacement and refined to an R‐value of 17.9% at 2.0 Å. As a member of the short‐chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two‐step reaction mechanism is proposed for cis‐dihydrodiol dehydrogenases.
Structural investigation of the A domains of human blood coagulation factor V by molecular modeling
Authors: Villoutreix Bruno O. , Dahlbäck Björn
Published: February 2009

Factor V (FV) is a large (2,196 amino acids) nonenzymatic cofactor in the coagulation cascade with a domain Organization (A1‐A2‐B‐A3‐C1‐C2) similar to the one of factor VIII (FVIII). FV is activated to factor Va (FVa) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1‐A2) and a light chain (A3‐C1‐C2). Activated protein C (APC), together with its cofactor protein S (PS), inhibits the coagulation cascade via limited proteolysis of FVa and FVIIIa (APC cleaves FVa at residues R306, R506, and R679). The A domains of FV and FVIII share important sequence identity with the plasma copper‐binding protein ceruloplasmin (CP). The X‐ray structure of CP and theoretical models for FVIII have been recently reported. This information allowed us to build a theoretical model (994 residues) for the A domains of human FV/FVa (residues 1‐656 and 1546‐1883). Structural analysis of the FV model indicates that: (a) the three A domains are arranged in a triangular fashion as in the case of CP and the Organization of these domains should remain essentially the same before and after activation; (b) a Type II copper ion is located at the A1‐A3 interface; (c) residues R306 and R506 (cleavage sites for APC) are both solvent exposed; (d) residues 1667‐1765 within the A3 domain, expected to interact with the membrane, are essentially buried; (e) APC does not bind to FVa residues 1865‐1874. Several other features of factor V/Va, like the R506Q and A221V mutations; factor Xa (FXa) and human neutrophil elastase (HNE) cleavages; protein S, Prothrombin and FXa binding, are also investigated.
Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase
Authors: Jeffery Constance J. , Petsko Gregory A. , Ringe Dagmar , Barry Tom , Doonan Shawn
Published: February 2009

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 Å resolution in the presence of the cofactor pyridoxal‐5′‐phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R‐factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the “closed” conformation with a bound maleate inhibitor in each active site. It shares the same three‐dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.
Fold prediction by a hierarchy of sequence, threading, and modeling methods
Authors: Jaroszwski ŁUkasz , Rychlewski Leszek , Zhang Baohong , Godzik Adam
Published: February 2009

Several fold recognition algorithms are compared to each other in terms of prediction accuracy and significance. It is shown that on standard benchmarks, hybrid methods, which combine scoring based on sequence‐sequence and sequence‐structure matching, surpass both sequence and threading methods in the number of accurate predictions. However, the sequence similarity contributes most to the prediction accuracy. This strongly argues that most examples of apparently nonhomologous proteins with similar folds are actually related by evolution. While disappointing from the perspective of the fundamental understanding of protein folding, this adds a new significance to fold recognition methods as a possible first step in function prediction.

Despite hybrid methods being more accurate at fold prediction than either the sequence or threading methods, each of the methods is correct in some cases where others have failed. This partly reflects a different perspective on sequence/structure relationship embedded in various methods. To combine predictions from different methods, estimates of significance of predictions are made for all methods. With the help of such estimates, it is possible to develop a “jury” method, which has accuracy higher than any of the single methods. Finally, building full three‐dimensional models for all top predictions helps to eliminate possible false positives where alignments, which are optimal in the one‐dimensional sequences, lead to unsolvable sterical conflicts for the full three‐dimensional models.
In vivo acetylation identified at lysine 70 of human lens αA‐crystallin
Authors: Lin Peiping P. , Barry Richard C. , Smith David L. , Smith Jean B.
Published: February 2009

Posttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of αA‐crystallin has been isolated from the water‐soluble portion of human lenses. The αA‐crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of αA‐crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of αA‐crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of αA‐crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of αA‐crystallin.
Prediction of functional residues in water channels and related proteins
Authors: Froger A. , Thomas D. , Delamarche C. , Tallur B.
Published: February 2009

In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico‐chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport.
Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein
Authors: Albright Ronald A. , Matthews Brian W. , Mossing Michael C.
Published: February 2009

The structure has been determined at 3.0 Å resolution of a complex of engineered monomeric Cro repressor with a seven‐base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half‐operator that is recognized by each subunit of the wild‐type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence‐independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48° further out of the major groove, while the turn region of the helix‐turn‐helix remains in contact with the DNA backbone. All of the direct base‐specific interactions seen in the wild‐type Cro‐operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein‐DNA interface than for half of the wild‐type Cro‐operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence‐nonspecific interaction of the wild‐type Cro dimer with DNA. In the sequence‐specific complex, the DNA is bent, the protein dimer undergoes a large hinge‐bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.
Scan‐rate dependence in protein calorimetry: The reversible transitions of Bacillus circulans xylanase and a disulfide‐bridge mutant
Authors: Davoodi Jamshid , Wakarchuk Warren W. , Surewicz Witold K. , Carey Paul R.
Published: February 2009

The stabilities of Bucillus circuluns xylanase and a disulfide‐bridge‐containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild‐type protein showed a scan‐rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan‐rate dependent in the presence of urea than in the urea‐free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan‐rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706‐12712). The kinetic factors responsible for scan‐rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan‐rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide‐bridge‐containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate‐limiting step on the thermal denaturation pathway.
Engineering of betabellin‐15d: A 64 residue beta sheet protein that forms long narrow multimeric fibrils
Authors: Lim Amareth , Saderholm Matthew J. , Kroll Mathias , Yan Yibing , Perera Lalith , Erickson Bruce W. , Makhov Alexander M. , Griffith Jack D.
Published: February 2009

The betabellin target structure is a β‐sandwich protein consisting of two 32 residue β‐sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin‐15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p = DPrO, k = DLYS, and h = DHis) did not fold in water at pH 6.5. Air oxidation of betabellin‐15S provided betabellin‐15D, the 64 residue disulfide bridged two‐chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of β structure observed for betabellin‐15D increased with the pH and ionic strength of the solution and the betabellin‐15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin‐15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin‐15D fibrils are consistent with a single row of β‐sandwich molecules joined by multiple intersheet H‐bonds.
Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond
Authors: Ikeguchi Masamichi , Kato Masao , Sugai Shintaro , Fujino Motoaki , Kuwajima Kunihiro
Published: February 2009

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of α‐lactalbumin, in which the 6‐120 disulfide bond is selectively reduced and S‐carboxymethylated, was measured and compared with that of disulfide‐intact α‐lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate‐controlled folding model and the multiple‐pathway folding model, that we had proposed previously. All of the data supported the multiple‐pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of α‐lactalbumin is not an obligatory intermediate. The cleavage of the 6‐120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6‐120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross‐link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of α‐lactalbumin is discussed.
An improved chemical approach toward the C‐terminal sequence analysis of proteins containing all natural amino acids
Authors: Hardeman Klaas , Samyn Bart , Beeumen Jozef Van , Eycken Johan Van Der
Published: February 2009

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N‐acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H‐NMR. This chemical method allows, for the first time, complete derivatization of N‐acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C‐terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C‐terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C‐terminal amino acids including proline is a major step forward in the development of a general chemical C‐terminal sequencing method that permits the C‐terminal sequence analysis of proteins of any amino acid composition.
The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA
Authors: Chen Jiwen , Smith David L. , Griep Mark A.
Published: February 2009

Differential chemical modification of the lysines and amino‐terminus of Escherichia coli single‐strand binding (SSB) protein was used to determine their roles in the binding of SSB to single‐stranded DNA (ssDNA). A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride. First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single‐stranded ssDNA. Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride. Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry. The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA. Acetylation rate constants were determined from time course measurements. In the absence of ssDNA, the terminal α‐amine of SSB was I0‐fold more reactive than Lys residues at positions 43, 62, 73, and 87. The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition. In the presence of ssDNA, the reactivities of the amino‐terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7‐25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA. Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer‐monomer interface.
Class‐directed structure determination: Foundation for a protein structure initiative
Authors: Terwilliger Thomas C. , Waldo Geoffrey , Peat Thomas S. , Newman Janet M. , And Kelvin Chu , Berendzen Joel
Published: February 2009

The recent sequencing of many complete genomes, combined with the development of methods that allow rapid structure determination for many proteins, has changed the way in which protein structure determinations can be approached. One‐by‐one determinations of individual protein structures will soon be augmented by class‐directed structure analyses in which a group of proteins is targeted and structures of representative members are determined and used to represent the entire group. Such a shift in approach would be the foundation for a broad protein structure initiative targeting classes of proteins important for biotechnology and for a fundamental understanding of protein function.
Conversion of a β‐strand to anα‐helix induced by a single‐site mutation observed in the crystal structure of fis mutant pro26Ala
Authors: Yang Wei‐Zen , Ko Tzu‐Ping , Yuan Hanna S. , Corselli Leah , Johnson Reid C.
Published: February 2009

The conversion from anα‐helix to a β‐strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self‐assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a β‐strand to anα‐helix by a single‐site mutation as observed in the crystal structure of Fis mutant Pro26 Ala determined at 2.0 Å resolution. Pro26 in Fis occurs at the point where a flexible extended β‐hairpin arm leaves the core structure. Thus it can be classified as a “hinge proline” located at the C‐terminal end of the β2‐strand and the N‐terminal cap of the Aα‐helix. The replacement of Pro26 to alanine extends the Aα‐helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted fromα β‐strand to anα‐helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin‐catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its β‐strand conformation in the crystal; therefore, this peptide shows a “chameleon‐like” character since it can adopt either anα‐helix or a β‐strand structure in different environments. The structure of Pro26 Ala provides an additional example where not only the protein sequence, but also non‐local interactions determine the secondary structure of proteins.