bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling of protein loops by simulated annealing
Authors: Collura V. , Higo Junichi , Garnier J.
Published: February 2009

A method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33–43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin. The predicted structure is obtained from the ensemble average of the coordinates of the Monte Carlo simulation at 300 K, which exhibits the lowest internal energy. The starting conformation of the loop prior to modeling is chosen to be completely extended, and a closing harmonic potential is applied to N, CA, C, and O atoms of the terminal residues. A rigid geometry potential of Robson and Platt (1986, J. Mol. Biol. 188, 259–281) with a united atom representation is used. This we demonstrate to yield a loop structure with good hydrogen bonding and torsion angles in the allowed regions of the Ramachandran map. The average accuracy of the modeling evaluated on the eight modeled loops is 1 Å root mean square deviation (rmsd) for the backbone atoms and 2.3 Å rmsd for all heavy atoms.
Confidence limits on the branching order of phylogenetic trees
Authors: Shearer Andrew C. , Johnson Mark S.
Published: February 2009

We describe a confidence test for branching order that can aid protein phylogeny reconstruction as well as the evaluation of the optimal tree. It is proposed that the process resulting in the observed amino acid residue differences, which is the basis for the identification of the order and relative times of divergence events, is appropriately described by a modification of the negative binomial distribution. The relative total numbers of mutations (accepted and nonaccepted), which result in a given number of amino acid differences, may be obtained as the expectation of this distribution. The associated variances enable significant differences in tree branching order to be established. If the total rates of mutation of the genes encoding the compared proteins are equal, the expected total mutations and their associated variances map identically to their relative times of divergence. In addition, significantly different rates of change (due to differences in total mutation rate and/or acceptance rate) may be identified without the requirement of an outlying reference group. The method is equally applicable to phylogenies derived from DNA or RNA sequence information.
Calculation of protein backbone geometry from α‐carbon coordinates based on peptide‐group dipole alignment
Authors: Liwo A. , Wawak R. J. , Scheraga H. A. , Pincus M. R. , Rackovsky S.
Published: February 2009

An algorithm is proposed for the conversion of a virtual‐bond polypeptide chain (connected Cα atoms) to an all‐atom backbone, based on determining the most extensive hydrogen‐bond network between the peptide groups of the backbone, while maintaining all of the backbone atoms in energetically feasible conformations. Hydrogen bonding is represented by aligning the peptide‐group dipoles. These peptide groups are not contiguous in the amino acid sequence. The first dipoles to be aligned are those that are both sufficiently close in space to be arranged in approximately linear arrays termed dipole paths. The criteria used in the construction of dipole paths are: to assure good alignment of the greatest possible number of dipoles that are close in space; to optimize the electrostatic interactions between the dipoles that belong to different paths close in space; and to avoid locally unfavorable amino acid residue conformations. The equations for dipole alignment are solved separately for each path, and then the remaining single dipoles are aligned optimally with the electrostatic field from the dipoles that belong to the dipole‐path network. A least‐squares minimizer is used to keep the geometry of the α‐carbon trace of the resulting backbone close to that of the input virtual‐bond chain. This procedure is sufficient to convert the virtual‐bond chain to a real chain; in applications to real systems, however, the final structure is obtained by minimizing the total ECEPP/2 (empirical conformational energy program for peptides) energy of the system, starting from the geometry resulting from the solution of the alignment equations. When applied to model α‐helical and β‐sheet structures, the algorithm, followed by the ECEPP/2 energy minimization, resulted in an energy and backbone geometry characteristic of these α‐helical and β‐sheet structures. Application to the α‐carbon trace of the backbone of the crystallographic 5PTI structure of bovine pancreatic trypsin inhibitor, followed by ECEPP/2 energy minimization with C
α
‐distance constraints, led to a structure with almost as low energy and root mean square deviation as the ECEPP/2 geometry analog of 5PTI, the best agreement between the crystal and reconstructed backbone being observed for the residues involved in the dipole‐path network.
Comparison of conformational characteristics in structurally similar protein pairs
Authors: Flores T.P. , Orengo C.A. , Moss D.S. , Thornton J.M.
Published: February 2009

Although it is known that three‐dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main‐chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of Cα coordinates but also accessibility, Ooi numbers, secondary structure, and side‐chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically.
A peptide analog of the calmodulin‐binding domain of myosin light chain kinase adopts an aL‐helical structure in aqueous trifluoroethanol
Authors: Zhang Mingjie , Yuan Tao , Vogel Hans J.
Published: February 2009

A 22‐residue synthetic peptide encompassing the calmodulin (CaM)‐binding domain of skeletal muscle myosin light chain kinase was studied by two‐dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix‐inducing solvent trifluoroethanol (TFE) causes it to form an α‐helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum‐filtered J‐correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the α‐carbon chemical shifts were obtained from (1H,13C)‐heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), dαN( i, i + 3), and d αβ (i, i + 3) NOE crosspeaks indicates that an α ‐helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield α ‐proton and downfield α ‐carbon shifts in this region of the peptide provide further support for the formation of an α ‐helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.
A 19F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli
Authors: Sun Zhen‐Yu , Truong Hoai‐Thu N. , Pratt E. A. , Sutherland David C. , Kulig Christine E. , Homer Ronald J. , Groetsch Stefan M. , Hsue Priscilla Y. , Ho Chien
Published: February 2009

d‐Lactate dehydrogenase (d‐LDH) is a membrane‐associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65, 000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of d‐LDH and its interaction with phospholipids. We incorporated 5‐fluorotryptophan (5F‐Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide‐directed mutagenesis, and studied the 5F‐Trp‐labeled enzymes using 19F‐NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin‐labeled fatty acid, which broadens the resonance from any residue within 15 Å, we have established that the membrane‐binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F‐NMR spectroscopy of wild‐type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin‐labeled fatty acid. These experiments indicate that 9–10 Phe and 3–4 Tyr residues are located near the lipid phase.
Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation
Authors: Thomas Paul D. , Dill Ken A.
Published: February 2009

How important are helical propensities in determining the conformations of globular proteins? Using the two‐dimensional lattice model and two monomer types, H (hydrophobic) and P (polar), we explore both nonlocal interactions, through an HH contact energy, as developed in earlier work, and local interactions, through a helix energy, σ. By computer enumeration, the partition functions for short chains are obtained without approximation for the full range of both types of energy. When nonlocal interactions dominate, some sequences undergo coil‐globule collapse to a unique native structure. When local interactions dominate, all sequences undergo helix–coil transitions. For two different conformational properties, the closest correspondence between the lattice model and proteins in the Protein Data Bank is obtained if the model local interactions are made small compared to the HH contact interaction, suggesting that helical propensities may be only weak determinants of globular protein structures in water. For some HP sequences, varying σ/ leads to additional sharp transitions (sometimes several) and to “conformational switching” between unique conformations. This behavior resembles the transitions of globular proteins in water to helical states in alcohols. In particular, comparison with experiments shows that whereas urea as a denaturant is best modeled as weakening both local and nonlocal interactions, trifluoroethanol is best modeled as mainly weakening HH interactions and slightly enhancing local helical interactions.
Identification of the amino acids comprising a surface‐exposed epitope within the nucleotide‐binding domain of the na+, K+‐ATPase using a random peptide library
Authors: Malik Bela , Jamieson Gordon A. , Ball W. James
Published: February 2009

Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8‐P1‐A3 has been shown to bind to the catalytic (α) subunit of the Na+, K+‐ATPase holoenzyme and the synthetic peptide sequence 496‐HLLVMK*GAPER‐506, which includes Lys 501 (K*), the major site for fluorescein‐5′‐isothio‐cyanate labeling of the Na+, K+‐ATPase. This sequence region of α is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W.R. & Green, N.W., 1989, Eur. J. Biochem. 179, 241–248). In this study we have determined M8‐P1‐A3′s ability to recognize the α‐subunit or homologous E1E2‐ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or “epitope” library, recently developed by Scott and Smith (1990, Science 249, 386–390) and Devlin et al. (Devlin, J.J., Panganiban, L.C., & Devlin, P.E., 1990, Science 249, 404–406), has served as a convenient technique to confirm the species‐specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8‐P1‐A3 epitope was found to consist of the five amino acid 494–PRHLL‐498 sequence stretch of α, with residues PRxLx being critical for antibody recognition.
Origins of structural diversity within sequentially identical hexapeptides
Authors: Cohen Bruce I. , Presnell Scott R. , Cohen Fred E.
Published: February 2009

Efforts to predict protein secondary structure have been hampered by the apparent structural plasticity of local amino acid sequences. Kabsch and Sander (1984, Proc. Natl. Acad. Sci. USA 81, 1075–1078) articulated this problem by demonstrating that identical pentapeptide sequences can adopt distinct structures in different proteins. With the increased size of the protein structure database and the availability of new methods to characterize structural environments, we revisit this observation of structural plasticity. Within a set of proteins with less than 50% sequence identity, 59 pairs of identical hexapeptide sequences were identified. These local structures were compared and their surrounding structural environments examined. Within a protein structural class (α/α, β/β, α/β, α + β), the structural similarity of sequentially identical hexapeptides usually is preserved. This study finds eight pairs of identical hexapeptide sequences that adopt β‐strand structure in one protein and α‐helical structure in the other. In none of the eight cases do the members of these sequence pairs come from proteins within the same folding class. These results have implications for class dependent secondary structure prediction algorithms.
Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability
Authors: Pjura Philip , Matsumura Masazumi , Baase Walter A. , Matthews Brian W.
Published: February 2009

An M13 bacteriophage‐based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates. Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one‐step mutagenesis and screening system has been used to find 18 random single‐site mutant lysozymes, of which 11 were heat resistant. Each of these had a melting temperature within 0.8–1.4°C of wild type, suggesting that the screening system is quite sensitive.
Side‐chain entropy and packing in proteins
Authors: Bromberg Sarina , Dill Ken A.
Published: February 2009

What role does side‐chain packing play in protein stability and structure? To address this question, we compare a lattice model with side chains (SCM) to a linear lattice model without side chains (LCM). Self‐avoiding configurations are enumerated in 2 and 3 dimensions exhaustively for short chains and by Monte Carlo sampling for chains up to 50 main‐chain monomers long. This comparison shows that (1) side‐chain degrees of freedom increase the entropy of open conformations, but side‐chain steric exclusion decreases the entropy of compact conformations, thus producing a substantial entropy that opposes folding; (2) there is a side‐chain “freezing” or ordering, i.e., a sharp decrease in entropy, near maximum compactness; and (3) the different types of contacts among side chains (s) and main‐chain elements (m) have different frequencies, and the frequencies have different dependencies on compactness.mmcontacts contribute significantly only at high densities, suggesting that main‐chain hydrogen bonding in proteins may be promoted by compactness. The distributions ofmm, ms, andsscontacts in compact SCM configurations are similar to the distributions in protein structures in the Brookhaven Protein Data Bank. We propose that packing in proteins is more like the packing of nuts and bolts in a jar than like the pairwise matching of jigsaw puzzle pieces.
Relative stabilities of synthetic peptide homo‐ and heterodimeric troponin‐C domains
Authors: Shaw Gary S. , Hodges Robert S. , Kay Cyril M. , Sykes Brian D.
Published: February 2009

It has previously been shown that synthetic peptides corresponding to calcium‐binding sites III (SCIII) and IV (SCIV) from troponin‐C can undergo a calcium‐induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280–283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258–6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer (Shaw GS et al., 1992a, J Am Chem Soc 114:6258–6259). The stabilities of these dimers have been investigated by using 1H‐NMR and circular dichroism spectroscopies to follow temperature‐ and guanidine hydrochloride (GuHCl)‐induced denaturations. It has been found that the most stable species, the SCIII/SCIV heterodimer (δGu = –64.8 kJ/mol), is about 13 kJ/mol more stable than the least stable species, the SCIV homodimer, while the SCIII homodimer is of intermediate stability. This trend of free energies agrees well with the trend of δG0 values derived from the products of the dissociation constants for calcium binding and peptide association determined from earlier calcium‐titration studies. These observations provide evidence that calcium affinity and the association of 2‐calcium binding sites are tightly linked. However, it was noted that in all cases δG0 was considerably more negative than δGu determined from GuHCl experiments. This difference increased as the stability of the peptide complex increased, providing evidence that linear extrapolation of GuHCl data for very stable proteins may significantly underestimate the value for δG0.
Interaction of the DNA‐binding domain of Drosophila heat shock factor with its cognate DNA site: A thermodynamic analysis using analytical ultracentrifugation
Authors: Kim Soon‐Jong , Tsukiyama Toshio , Lewis Marc S. , Wu Carl
Published: February 2009

Heat shock transcription factor (HSF) mediates the activation of heat shock genes by binding to its cognate sites with high affinity and specificity. The high‐affinity binding of HSF is dependent on the formation of an HSF homotrimer, which interacts specifically with the heat shock response element (HSE), comprised of 3 inverted repeats of the 5‐bp sequence NGAAN. In order to investigate the thermodynamic basis of the interaction between HSF and HSE, we have overexpressed and purified a polypeptide (dHSF(33–163)) encompassing only the DNA‐binding domain of HSF from Drosophila and analyzed its binding to DNA by equilibrium analytical ultracentrifugation using a multiwavelength scan technique. We demonstrate that dHSF(33–163) can bind as a monomer with 1:1 stoichiometry to a synthetic 13‐bp DNA containing a single NGAAN sequence. The values of the thermodynamic parameters obtained from the temperature dependence of the equilibrium binding constants indicate that the changes of free energy for the binding of dHSF(33–163) to the wild‐type site and a mutant DNA site are predominantly characterized by substantial negative changes of enthalpy. Binding to the wild‐type DNA is characterized by a significant positive change of entropy, whereas binding to the mutant DNA is distinguished by a negative change of entropy of comparable magnitude. The binding to the mutant DNA was also highly sensitive to increasing salt concentrations, indicating a dominance of ionic interactions. The sequence‐specific, 1:1 binding of dHSF(33–163) to the NGAAN sequence provides a basis for the analysis of higher order interactions between HSF trimers and the HSE.
UDP‐glucose dehydrogenase from bovine liver: Primary structure and relationship to other dehydrogenases
Authors: Hempel John , Perozich John , Romovacek Hana , Hinich Amy , Kuo Ingrid , Feingold David Sidney
Published: February 2009

The primary structure of bovine liver UDP‐glucose dehydrogenase (UDPGDH), a hexameric, NAD+‐linked enzyme, has been determined at the protein level. The 52‐kDa subunits are composed of 468 amino acid residues, with a free N‐terminus and a Ser/Asn microheterogeneity at one position. The sequence shares 29.6% positional identity with GDP‐mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to “UDP‐N‐acetyl‐mannosaminuronic acid dehydrogenase” from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of ∼26–34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4‐electron‐transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half‐reaction (conversion of UDP‐aldehydoglucose to UDP‐glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half‐reaction (conversion of UDP‐glucose to UDP‐aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme‐binding fold is found at positions 11–16, close to the N‐terminus as with “short‐chain” alcohol dehydrogenases. Because the enzyme combines functionalities of alcohol and aldehyde dehydrogenases, it was also of interest to search specifically for other sequence similarities to either of these 2 enzymes, as well as to histidinol dehydrogenase, another 4‐electron‐transferring dehydrogenase, but none were found.
Crystal structure of inorganic pyrophosphatase from Thermus thermophilus
Authors: Teplyakov Alexei , Obmolova Galya , Wilson Keith S. , Ishii Keisuke , Kaji Hiroyuki , Samejima Tatsuya , Kuranova Inna
Published: February 2009

The 3‐dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T‐PPase) has been determined by X‐ray diffraction at 2.0 Å resolution and refined to R = 15.3%. The structure consists of an antiparallel closed β‐sheet and 2 α‐helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek‐key topology of the 5‐stranded β‐barrel was found in the OB‐fold proteins, the bacteriophage gene‐5 DNA‐binding protein, toxic‐shock syndrome toxin‐1, and the major cold‐shock protein of Bacillus subtilis. Moreover, all known nucleotide‐binding sites in these proteins are located on the same side of the β‐barrel as the active center in T‐PPase. Analysis of the active center of T‐PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site‐directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T‐PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.
The pharmacophore of the human C5a anaphylatoxin
Authors: Toth Matthew J. , Huwyler Leslie , Boyar William C. , Braunwalder Albert F. , Yarwood Donna , Hadala Joseph , Haston William O. , Sills Matthew A. , Seligmann Bruce , Galakatos Nicholas
Published: February 2009

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74‐amino acid protein was performed and the effects on the potency of receptor binding and of C5a‐induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site‐directed mutagenesis. Ten noncontiguous amino acids from the structurally well‐defined 4‐helix core domain (amino acids 1–63) and the C‐terminal arginine‐containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C‐terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.
Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals
Authors: Duke E.M.H. , Wakatsuki S. , Hadfield A. , Johnson L.N.
Published: February 2009

The conversion of substrate, heptenitol, to product, β‐1‐C‐methyl, α‐D‐glucose‐1‐phosphate (heptulose‐2‐P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5‐dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer.

In the Laue diffraction experiments, data to 2.8 Å resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800‐ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed.

Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 Å resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose‐2‐P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 Å resolution to establish the fractional occupancies of the enzyme‐substrate and enzyme‐product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal. In these experiments in which only partial reaction had occurred, there was connecting density between the phosphate and the O2 hydroxyl of the sugar substrate. The recent evidence for the influence of the O2 hydroxyl on the catalytic mechanism is discussed.
Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala
Authors: De Llano Jose Javier Martin , Manning James M.
Published: February 2009

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(β), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val‐6(β) of one tetramer and the hydrophobic region between Phe‐85(β) and Leu‐88(β) on an adjacent tetramer. Thus, a construct encoding the donor Val‐6(β) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu‐88(β) was assembled. The doubly mutated β‐globin gene was expressed in yeast together with the normal human α‐chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant β‐chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor‐acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.
Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine
Authors: Yanase Hideshi , Cahill Sean , De Llano Jose Javier Martin , Manning Lois R. , Schneider Klaus , Chait Brian T. , Vandegriff Kim D. , Winslow Robert M. , Manning James M.
Published: February 2009

Site‐directed mutagenesis of an important subunit contact site, Asp‐99(β), by a Lys residue (D99K(β)) was proven by sequencing the entire β‐globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2–15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3‐diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(β) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the α‐ and β‐globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(β) recombinant mutant forms have differences in their heme‐protein environments.
Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase
Authors: Tominaga Nobuaki , Jameson David M. , Uyeda Kosaku
Published: February 2009

Reversible unfolding of rat testis fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2‐(iodoacetyl)amino) ethyl)naphthalene‐1‐sulfonic acid) probes. The unfolding reaction is described minimally as a 4‐state transition from folded dimer → partially unfolded dimer → monomer → unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp‐15 in the N‐terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6‐phosphate, but not ATP or fructose 2,6‐bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N‐terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.
Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem‐5
Authors: Lim Wendell A. , Fox Robert O. , Richards Frederic M.
Published: February 2009

We have determined the thermodynamic stability and peptide binding affinity of the carboxy‐terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal‐transduction protein Sem‐5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation — at pH 7.3, the protein has a Tm of 73.1 °C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm ≅ 80 °C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more car‐boxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 °C. We have also characterized binding of the domain to 2 different length proline‐rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem‐5′s likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2‐fold increase in fluorescence intensity. Both bind with only modest affinities (Kd ≅ 30 μM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly‐L‐proline in a similar fashion.
Two‐step selective formation of three disulfide bridges in the synthesis of the C‐terminal epidermal growth factor‐like domain in human blood coagulation factor IX
Authors: Yang Yan , Sweeney William V. , Schneider Klaus , Chait Brian T. , Tam James P.
Published: February 2009

The 45‐residue C‐terminal EGF‐like domain in human blood coagulation factor IX has been synthesized by a 2‐step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4‐methyl‐benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1–3), Cys12 to Cys26 (Cys 2–4), or Cys28 to Cys41 (Cys 5–6). Only the peptide having the Cys 2–4 pair blocked with Acm forms the desired disulfide isomer (Cys 1–3/5–6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF‐like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.
A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA
Authors: Guenot Jeanmarie , Fletterick Robert J. , Kollman Peter A.
Published: February 2009

The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan‐analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding‐site surface, a consequence of the tryptophan‐or tryptophan analogue‐induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein‐DNA complex. The effects of charge‐change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein‐DNA binding constants. A consistent model is formed that defines a role for long‐range electrostatics early in the protein‐DNA association process and complements previous structural, molecular association, and mutagenesis studies.
The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability
Authors: Völkl Paul , Markiewicz Peter , Stetter Karl O. , Miller Jeffrey H.
Published: February 2009

The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 °C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80–130 °C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named “aerolysin”) was determined. Multiple sequence alignment with 15 different serine‐type proteases showed greatest homology with subtilisins from gram‐positive bacteria rather than archaeal or eukaryal serine proteases.

Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha‐helices. Aspartic acids were found at the N‐terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
Extending the B7 (CD80) gene family
Authors: Linsley Peter S. , Peach Robert , Gladstone Paul , Bajorath Jürgen
Published: February 2009

B7‐1 and B7‐2 are members of the immunoglobulin superfamily (IgSF) and important regulators of T cell‐mediated immune responses. Despite sharing only limited sequence identity, B7‐1 and B7‐2 bind common receptors, CD28 and CTLA‐4, on T cells and have similar functional properties. We have found that the extracellular V(ariable)‐like domains of B7‐1 and B7‐2 share significant sequence similarities with 3 major histocompatibility complex (MHC)‐encoded members of the IgSF: butyrophilin, myelin/oligodendrocyte glycoprotein, and the chicken MHC molecule, B‐G. This raises the question whether there is an evolutionary link between the MHC, which encodes molecules regulating the antigen specificity of T lymphocyte responses, and B7 molecules, which co‐stimulate these responses in antigen‐nonspecific fashion.
Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores
Authors: Wimley William C. , Selsted Michael E. , White Stephen H.
Published: February 2009

Defensins comprise a family of broad‐spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP‐2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP‐2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP‐2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP‐2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP‐2, and the POPG:POPC ratio. Vesicles (300 μM POPG) readily aggregated at HNP‐2 concentrations above 1 μM, but no mixing of vesicle contents could be detected for concentrations as high as 2 μM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi‐fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 μM POPG vesicles, 50% of entrapped solute is released by 0.7 μM HNP‐2. We introduce a simple method for determining whether leakage from vesicles is graded or all‐or‐none. We show by means of this fluorescence “requenching” method that native HNP‐2 induces vesicle leakage in an all‐or‐none manner, whereas reduced HNP‐2 induces partial, or graded, leakage of vesicle contents. At HNP‐2 concentrations that release 100% of small (∼400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900‐Da dextran is mostly retained. These results suggest that HNP‐2 can form pores that have a maximum diameter of approximately 25 Å. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.
Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability
Authors: Lebowitz Jacob , Kar Sambit , Braswell Emory , Mcpherson Sylvia , Richard Dean L.
Published: February 2009

Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus‐1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer‐dimer‐trimer and monomer‐dimer‐tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 °C, with a dimerization Ka value of 5.2 × 105 M−1 for both models. Ka values of 2.1 × 105 and 3.8 × 105 M−1 were obtained for the respective association models at 20 °C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 °C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 °C to 37 °C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.
Dimerization of βB2‐crystallin: The role of the linker peptide and the N‐ and C‐terminal extensions
Authors: Trinkl Sabine , Glockshuber Rudi , Jaenicke Rainer
Published: February 2009

βB2‐ and γB‐crystallins of vertebrate eye lens are 2‐domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of βB2‐ and γB‐domains are very similar, γB‐crystallin is always monomeric, whereas βB2‐crystallin associates to homodimers. It has been suggested that the linker or the protruding N‐ and C‐terminal arms of βB2‐crystallin (not present in γB) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two βB2 mutants. In the first mutant, the linker peptide was replaced with the one from γB (βB2γL). In the second mutant, the N‐ and C‐terminal arms of 15‐ and 12‐residues length were deleted (βB2ΔNC). The βB2γL mutant is monomeric, whereas the βB2ΔNC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the βh2 mutants, as well as their stabilities against denaturants, resemble those of wild‐type βB2‐crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in βB2‐crystallin is necessary for dimerization, whereas the N‐ and C‐terminal arms appear to be involved in preventing the formation of higher homo‐oligomers.
Equilibrium unfolding of Escherichia coli ribonuclease H: Characterization of a partially folded state
Authors: Dabora Jonathan M. , Marqusee Susan
Published: February 2009

We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild‐type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2‐state process. Acid denaturation, however, reveals a cooperative transition at ∼pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near‐UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1‐anilinonaphthalene, 8‐sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.