bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry
Authors: Papac Damon I. , Hoyes John , Tomer Kenneth B.
Published: February 2009

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen‐antibody complex followed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI/TOF). A mouse anti‐bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin‐releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen‐antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen‐antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed‐phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti‐bombesin monoclonal antibody was shown to comprise the last 7–8 residues (HWAVGHLM‐NH2) of GRP.
Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa
Authors: Norin Martin , Haeffner Fredrik , Achour Adnane , Norin Torbjörn , Hult Karl
Published: February 2009

The substrate‐binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme.

Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme‐substrate transition‐state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.
Anatomy of an engineered NAD‐binding site
Authors: Mittl P.R.E. , Berry A. , Scrutton N. S. , Perham R. N. , Schulz G. E.
Published: February 2009

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2′‐phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38–43). In order to analyze the structural changes involved, we have determined 4 high‐resolution crystal structures, i.e., the structures of the wild‐type enzyme (1.86 Å resolution, R‐factor of 16.8%), of the wild‐type enzyme ligated with NADP (2.0 Å, 20.8%), of the NAD‐dependent mutant (1.74 Å, 16.8%), and of the NAD‐dependent mutant ligated with NAD (2.2 Å, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD‐binding site assumes a less rigid structure than the NADP site of the wild‐type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 Å from the sites of the mutations, this observation reminds us that mutations may have important long‐range consequences that are difficult to anticipate.
Structure comparison of the pheromones Er‐1, Er‐10, and Er‐2 from Euplotes raikovi
Authors: Luginbühl Peter , Ottiger Marcel , Mronga Siggi , WÜthrich Kurt
Published: February 2009

The NMR structures of the homologous pheromones Er‐1, Er‐10, and Er‐2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3‐helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half‐cystine residues in the individual sequences within the preserved 3‐dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er‐2, which is accommodated by a transition from an α‐helix in Er‐1 and Er‐10 to a 310‐helix in Er‐2. The most significant structural differences are located in the C‐terminal part of the proteins, which may have an important role in specific receptor recognition.
Identification of a molecular switch that selects between two crystal forms of bovine pancreatic trypsin inhibitor
Authors: Gallagher Warren H. , Croker Kevin M.
Published: February 2009

Two crystal forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2‐fold axis in form III crystals, the Lys 41 side‐chain amino nitrogens from 2 symmetry‐related molecules are only 2.72 Å apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side‐chain amino group is protonated and forms a salt bridge with a solvent‐derived phosphate group. For the Lys 41 side‐chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional inter‐molecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals.
Polar zippers: Their role in human disease
Authors: Perutz Max
Published: February 2009

Ascaris hemoglobin consists of 8 subunits, each of which contains a C‐terminal peptide with the sequence Glu‐Glu‐Lys‐His repeated 4 times. When plotted on a β‐strand, this sequence leads to alternate lysines and glutamates on one side of the strand, and alternate glutamates and histidines on the other side, suggestive of a polar zipper that links the subunits together. A computer search of the protein database showed that the same or similar sequences also occur in other proteins. Some contain long repeats of Asp‐Arg or Glu‐Arg, among them the small nuclear ribonucleo‐U1 70K protein, which is an autoantigen in systemic lupus erythematosis. These repeats appear to constitute the dominant epitopes in the autoimmune reaction. Single chains with Asp‐Arg repeats may form α‐helices in which alternate positively charged ridges and negatively charged grooves compensate each other. Several separate chains with Asp‐Arg repeats could compensate each other's charges optimally by zipping together to β‐sheets.

Several homeodomains of Drosophila, as well as the human transcription factor SP1, contain repeats of glutamines. Molecular modeling, circular dichroism, and electron and X‐ray diffraction studies of a synthetic poly(L‐glutamine) showed that it forms β‐sheets held together by hydrogen bonds between the main‐chain and side‐chain amides. Published data suggest that the function of these glutamine repeats consists of joining essential transcription factors bound to distant segments of DNA.

The study of the structure and function of glutamine repeats has assumed medical importance with the discovery that Huntington's disease and 3 other dominantly inherited diseases are associated with a lengthening of glutamine repeats in the proteins coded for by the affected genes.
Control of nucleation of protein crystals
Authors: Blow D.M. , Chayen N.E. , Lloyd L.F. , Saridakis E.
Published: February 2009

Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogeneous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase G2. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.
Protein crystallography and infectious diseases
Authors: Verlinde Christophe L.M.J. , Merritt Ethan A. , Van Den Akker Focco , Kim Hidong , Feil Ingeborg , Delboni Luis F. , Mande Shekhar C. , Sarfaty Steve , Petra Philip H. , Hol Wim G.J.
Published: February 2009

The current rapid growth in the number of known 3‐dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man.

We present here 2 illustrations of structure‐based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand‐binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti‐cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat‐labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity.

Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure‐based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.
Influenza virus neuraminidase: Structure, antibodies, and inhibitors
Authors: Colman P.M.
Published: February 2009

The determination of the 3‐dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti‐influenza virus drug.
Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors
Authors: Kossiakoff Anthony A. , Somers William , Ultsch Mark , Andow Kerrie , Muller Yves A. , De Vos Abraham M.
Published: February 2009

The crystal structures of complexes of human growth hormone (hGH) with the growth hormone and prolactin receptors (hGHR and hPRLR, respectively), together with the mutational data available for these systems, suggest that an extraordinary combination of conformational adaptability, together with finely tuned specificity, governs the molecular recognition processes operative in these systems. On the one hand, in the active 1:2 ligand‐receptor complexes, 2 copies of the same receptor use the identical set of binding determinants to recognize topographically different surfaces on the hormone. On the other hand, comparing the 1:1 hGH‐hGHR and hGH‐hPRLR complexes, 2 distinct receptors use this same set of binding determinants to interact with the identical binding site on the ligand, even though few residues among the binding determinants are conserved. The structural evidence demonstrates that this versatility is accomplished by local conformational flexibility of the binding loops, allowing adaptation to different binding environments, together with rigid‐body movements of the receptor domains, necessary for the creation of specific interactions with the same binding site.
Quantification of tertiary structural conservation despite primary sequence drift in the globin fold
Authors: Aronson Hans‐Erik G. , Hendrickson Wayne A. , Royer William E.
Published: February 2009

The globin family of protein structures was the first for which it was recognized that tertiary structure can be highly conserved even when primary sequences have diverged to a virtually undetectable level of similarity. This principle of structural inertia in molecular evolution is now evident for many other protein families. We have performed a systematic comparison of the sequences and structures of 6 representative hemoglobin subunits as diverse in origin as plants, clams, and humans. Our analysis is based on a 97‐residue helical core in common to all 6 structures. Amino acid sequence identities range from 12.4% to 42.3% in pairwise comparisons, and, despite these variations, the maximal RMS deviation in α‐carbon positions is 3.02 Å. Overall, sequence similarity and structural deviation are significantly anticorrelated, with a correlation coefficient of —0.71, but for a set of structures having under 20% pairwise identity, this anticorrelation falls to —0.38, which emphasizes the weak connection between a specific sequence and the tertiary fold. There is substantial variability in structure outside the helical core, and functional characteristics of these globins also differ appreciably. Nevertheless, despite variations in detail that the sequence dissimilarities and functional differences imply, the core structures of these globins remain remarkably preserved.
Electrostatic effects in the control of glycogen phosphorylase by phosphorylation
Authors: Johnson L.N. , Barford D.
Published: February 2009

Electrostatic effects are important in the initial activation mechanism of glycogen phosphorylase by phosphorylation. Analysis of the electrostatic surface potential of glycogen phosphorylase with the program GRASP shows that in the unphosphorylated state, the N‐terminal 20 residues, which include a number of basic amino acids, are located close to a position on the surface of the molecule that is highly acidic. Upon phosphorylation by phosphorylase kinase at Ser 14, the N‐terminal residues change their position and conformation so that the Ser‐P is directed away from the acidic patch and to an intersubunit site where 2 arginines bind the phosphate. This recognition site is created through tertiary and quaternary structural changes that accompany the activation mechanism.
Structure‐function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice
Authors: Chao Heman , DeLuca Carl I. , Davies Peter L. , Sykes Brian D. , Sönnichsen Frank D.
Published: February 2009

Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice‐binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66‐amino‐acid‐long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 β‐strands are paired to form 2 triple‐stranded antiparallel sheets and 1 double‐stranded anti‐parallel sheet, with the 2 triple sheets arranged as an orthogonal β‐sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154–1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface‐accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple‐stranded sheet near the C‐terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild‐type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild‐type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2‐dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C‐terminal β‐sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP‐ice interaction.
Structural studies of the engrailed homeodomain
Authors: Clarke Neil D. , Kissinger Charles R. , Desjarlais John , Gilliland Gary L. , Pabo Carl O.
Published: February 2009

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R‐factor of 19.7% at a resolution of 2.1 Å. This structure offers a high‐resolution view of an important family of DNA‐binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8‐Å engrailed‐DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a “herringbone” packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1‐helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side‐chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51‐amino acid variant of engrailed (residues 6–56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full‐length domain.
Formation of a native‐like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor
Authors: Staley Jonathan P. , Kim Peter S.
Published: February 2009

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single‐disulfide intermediate [30–51] plays a key role. We have investigated a recombinant analog of [30–51] using 2‐dimensional nuclear magnetic resonance (2D‐NMR). This recombinant analog, named [30–51]Ala, contains a disulfide bond between Cys‐30 and Cys‐51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D‐NMR, [30–51]Ala consists of 2 regions —one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30–51], named PαPβ, that contains a native‐like subdomain with tertiary packing. The unfolded region includes the first 14 N‐terminal residues of [30–51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30–51]Ala are structurally independent. The partially folded structure of [30–51]Ala explains many of the properties of authentic [30–51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30–51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native‐like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.
A common structural motif incorporating a cystine knot and a triple‐stranded β‐sheet in toxic and inhibitory polypeptides
Authors: Pallaghy Paul K. , Norton Raymond S. , Nielsen Katherine J. , Craik David J.
Published: February 2009

A common structural motif consisting of a cystine knot and a small triple‐stranded β‐sheet has been defined from comparison of the 3‐dimensional structures of the polypeptides ω‐conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI‐I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1–4 and 2–5) and the intervening polypeptide backbone, through which the third disulfide (3–6) passes. The other component of this motif is a triple‐stranded, anti‐parallel β‐sheet containing a minimum of 10 residues, XXC2, XC5X, XXC6X (where the numbers on the half‐cystine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cystine knot and antiparallel β‐sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.
Three‐dimensional model and quaternary structure of the human eye lens protein γS‐crystallin based on β‐ and γ‐crystallin X‐ray coordinates and ultracentrifugation
Authors: Zarina S. , Zaidi Z.H. , Driessen H. , Srinivasan N. , Slingsby C. , Jaenicke R.
Published: February 2009

A 3‐dimensional model of the human eye lens protein γS‐crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3‐dimensional structure of γ‐crystallin and β‐crystallin. The model is biased toward the monomeric γB‐crystallin, which is more similar in sequence. Bovine γS‐crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric γ‐crystallins. Because the linker has 1 extra residue compared with γB and βB2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. γS‐crystallin differs from γB‐crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both β‐ and γ‐crystallins, and may contribute to the different roles of these proteins in the lens.
Multiple protein structure alignment
Authors: Taylor William R. , Flores Tomas P. , Orengo Christine A.
Published: February 2009

A method was developed to compare protein structures and to combine them into a multiple structure consensus. Previous methods of multiple structure comparison have only concatenated pairwise alignments or produced a consensus structure by averaging coordinate sets. The current method is a fusion of the fast structure comparison program SSAP and the multiple sequence alignment program MULTAL. As in MULTAL, structures are progressively combined, producing intermediate consensus structures that are compared directly to each other and all remaining single structures. This leads to a hierarchic “condensation,” continually evaluated in the light of the emerging conserved core regions.

Following the SSAP approach, all interatomic vectors were retained with well‐conserved regions distinguished by coherent vector bundles (the structural equivalent of a conserved sequence position). Each bundle of vectors is summarized by a resultant, whereas vector coherence is captured in an error term, which is the only distinction between conserved and variable positions. Resultant vectors are used directly in the comparison, which is weighted by their error values, giving greater importance to the matching of conserved positions. The resultant vectors and their errors can also be used directly in molecular modeling.

Applications of the method were assessed by the quality of the resulting sequence alignments, phylogenetic tree construction, and databank scanning with the consensus. Visual assessment of the structural superpositions and consensus structure for various well‐characterized families confirmed that the consensus had identified a reasonable core.
A quantitative methodology for the de novo design of proteins
Authors: Brenner Steven E. , Berry Alan
Published: February 2009

We have developed a general quantitative methodology for designing proteins de novo, which automatically produces sequences for any given plausible protein structure. The method incorporates statistical information, a theoretical description of protein structure, and motifs described in the literature. A model system embodying a portion of the quantitative methodology has been used to design many protein sequences for the phage 434 Cro and fibronectin type III domain folds, as well as several other structures. Residue sequences selected by this prototype share no significant identity with any natural protein. Nonetheless, 3‐dimensional models of the designed sequences appear generally plausible. When examined using secondary structure prediction methods and profile analysis, the designed sequences generally score considerably better than the natural ones. The designed sequences are also in reasonable agreement with a sequence template. This quantitative methodology is likely to be capable of successfully designing new proteins and yielding fundamental insights about the determinants of protein structure.
Malate dehydrogenase: A model for structure, evolution, and catalysis
Authors: Goward Christopher R. , Nicholls David J.
Published: February 2009

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3‐dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3‐dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3‐dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.
Different protein sequences can give rise to highly similar folds through different stabilizing interactions
Authors: Laurents D. V. , Subbiah S. , Levitt M.
Published: February 2009

We report an interesting case of structural similarity between 2 small, nonhomologous proteins, the third domain of ovomucoid (ovomucoid) and the C‐terminal fragment of ribosomal L7/L12 protein (CTF). The region of similarity consists of a 3‐stranded β‐sheet and an α‐helix. This region is highly similar; the corresponding elements of secondary structure share a common topology, and the RMS difference for “equivalent” Cα atoms is 1.6 Å. Surprisingly, this common structure arises from completely different sequences. For the common core, the sequence identity is less than 3%, and there is neither significant sequence similarity nor similarity in the position or orientation of conserved hydrophobic residues. This superposition raises the question of how 2 entirely different sequences can produce an identical structure. Analyzing this common region in ovomucoid revealed that it is stabilized by disulfide bonds. In contrast, the corresponding structure in CTF is stabilized in the α‐helix by a composition of residues with high helix‐forming propensities. This result suggests that different sequences and different stabilizing interactions can produce an identical structure.
Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli
Authors: Georgiou George , Valax Pascal , Ostermeier Marc , Horowitz Paul M.
Published: February 2009

The enzyme TEM β‐lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM β‐lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0–1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis‐1‐anilino‐8‐naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM β‐lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0–1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM β‐lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM β‐lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131–136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.
Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C
Authors: Gagné Stéphane M. , Tsuda Sakae , Li Monica X. , Chandra Murali , Smillie Lawrence B. , Sykes Brian D.
Published: February 2009

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium‐loaded (1H, 15N, and 13C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical‐shift information, along with detailed NOE analysis and 3JHNHα coupling constants, permitted the determination and quantification of the Ca2+‐induced secondary structural change in the N‐domain of TnC. For both structures, 5 helices and 2 short β‐strands were found, as was observed in the apo N‐domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761–779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix‐B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca2+‐induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in α‐helical content associated with the transition from apo to the 2Ca2+ state of the N‐domain of TnC. Therefore, the Ca2+‐induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.
Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions
Authors: Monera Oscar D. , Kay Cyril M. , Hodges Robert S.
Published: February 2009

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled‐coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled‐coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of ⋍3.5 M) and, as well, their ΔΔGu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the ΔΔGu values correspondingly increased with the increasing differences in electrostatic interactions (20A – 15A5R, 1.5 kcal/mol; 20A – 10A10R, 3.7 kcal/mol; and 20A – 20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the uncharged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.
Tests for helix‐stabilizing interactions between various nonpolar side chains in alanine‐based peptides
Authors: Padmanabhan S. , Baldwin Robert L.
Published: February 2009

Straight‐chain, non‐natural, nonpolar amino acids norleucine, norvaline, and α‐amino‐n‐butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine‐based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix‐stabilizing i, i + 4 Tyr‐Leu and Tyr‐Val interactions reported earlier (Padmanabhan S, Baldwin RL, 1994, J Mol Biol 241:706–713). Leu spaced i, i + 4 from another Leu is measurably helix‐stabilizing relative to the corresponding i, i + 3 pair, but less so than for i, i + 4 Val‐Leu, Ile‐Leu, or Phe‐Leu pairs (relative to the corresponding i, i + 3 pairs) when Leu is C‐terminal to the other nonpolar amino acid. Our results indicate that limited side‐chain flexibility in an α‐helix strongly favors the interaction between 2 nonpolar residues to stabilize an isolated α‐helix.
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp‐369 → Asn): A mechanism involving one zinc per active site
Authors: Tibbitts Thomas T. , Xu Xu , Kantrowitz Evan R.
Published: February 2009

Using site‐directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp‐369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000‐fold lower than the value for the wild‐type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p‐nitrophenol phosphate was 2 orders of magnitude lower than for the wild‐type enzyme. Thus the kcal/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp‐369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 Å resolution with an R‐factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X‐ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
Redesigning the hydrophobic core of a four‐helix‐bundle protein
Authors: Munson Mary , Regan Lynne , O'Brien Ronan , Sturtevant Julian M.
Published: February 2009

Rationally redesigned variants of the 4‐helix‐bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native‐like physical properties of the wild‐type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild‐type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild‐type protein. The rate of chemically induced folding and unfolding of wild‐type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type.
Protein structural similarities predicted by a sequence‐structure compatibility method
Authors: Matsuo Yo , Nishikawa Ken
Published: February 2009

A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3‐dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP‐binding type A sequence motif and 2 ATP‐binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose‐binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N‐and C‐terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate‐binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase β subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.
Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: Absence of the conserved structural water
Authors: Kumar Amarendra , Sekharudu Chandra , Ramakrishnan Boopathy , Dupureur Cynthia M. , Zhu Hongxin , Tsai Ming‐Daw , Sundaralingam Muttaiya
Published: February 2009

To probe the role of the Asp‐99 → His‐48 pair in phospholipase A2 (PLA2) catalysis, the X‐ray structure and kinetic characterization of the mutant Asp‐99 → Asn‐99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3121 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801–11811). The 1.9‐Å X‐ray structure of the mutant showed that the carbonyl group of Asn‐99 side chain is hydrogen bonded to His‐48 in the same way as that of Asp‐99 in the WT, thus retaining the tautomeric form of His‐48 and the function of the enzyme. The NH2 group of Asn‐99 points away from His‐48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn‐102 is hydrogen bonded to His‐57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala‐1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn‐99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala‐1.
Probing protein‐cofactor interactions in the terminal oxidases by second derivative spectroscopy: Study of bacterial enzymes with cofactor substitutions and heme a model compounds
Authors: Felsch Jason S. , Horvath Martin P. , Gursky Susan , Hobaugh Michael R. , Goudreau Paul N. , Fee James A. , Morgan William T. , Admiraal Suzanne J. , Ikeda‐Saito Masao , Fujiwara Taketomo , Fukumori Yoshihiro , Yamanaka Tateo , Copeland Robert A.
Published: February 2009

Second derivative absorption spectra are reported for the aa3‐cytochrome c oxidase from bovine cardiac mitochondria, the aa3‐600 ubiquinol oxidase from Bacillus subtilis, the ba3‐cytochrome c oxidase from Thermus thermophilis, and the aco‐cytochrome c oxidase from Bacillus YN‐2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450‐nm absorption band of the terminal oxidases as the 6‐coordinate low‐spin heme, cytochrome a. The spectrum of the aco‐cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6‐coordinate low‐spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine‐proline‐rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A‐(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide‐inhibited mixed‐valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein‐cofactor interactions that are unique to the cytochrome a‐binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.
A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins
Authors: Watkins J. Abra , Cusanovich Michael A. , Meyer Terrance E. , Tollin Gordon
Published: February 2009

A “parallel plate” model describing the electrostatic potential energy of protein‐protein interactions is presented that provides an analytical representation of the effect of ionic strength on a bimolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site‐specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.
Effect of pH and phosphate ions on self‐association properties of the major cold‐shock protein from Bacillus subtilis
Authors: Makhatadze George I. , Marahiel Mohamed A.
Published: February 2009

The intermolecular interactions of the major cold‐shock protein from Bacillus subtilis (CspB) in solution in the presence of different salts, including phosphate, have been studied by means of scanning calorimetry and size‐exclusion chromatography. Calorimetric results indicate that, in all cases, protein unfolding can be approximated by a 2‐state model, but the modes of unfolding can differ depending on the conditions. In the presence of phosphate, the cooperative folding unit is a monomer, whereas in the absence of phosphate, the cooperative unit is a dimer. The difference in the self‐association of CspB in the presence and absence of phosphate was supported by size‐exclusion chromatography. These results are compared with recent structural studies of CspB in crystal and in solution.