bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: Theoretical clues for a plausible allosteric model of oxygen binding
Authors: Fariselli Piero , Bottoni Andrea , Bernardi Ferdinando , Casadio Rita
Published: February 2009

Abstract: In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruptus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.
Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin‐resistant boar seminal plasma O‐glycoprotein related to pB1 protein
Authors: Bezouška Karel , Sklenář Jan , Novák Petr , Halada Petr , Havlíček Vladimír , Kraus Marek , Tichá Marie , Jonáková VĚRa
Published: February 2009

The complete covalent structure of a novel boar DQH sperm surface protein resistant to many classical procedures of enzymatic fragmentation was determined. The relative molecular mass of the major form of this protein determined by ESI‐MS and MALDI‐MS was 13, 065.2 ±1.0 and 13, 065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N‐acetylhexosamine), 146 Da (i.e., plus deoxyhexose), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protein was determined using automated Edman degradation, MALDI‐MS, and post‐source decay (PSD) MALDI‐MS, and shown to consist of N‐terminal O‐glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O‐glycosidically linked to threonine 10, as confirmed by PSD MALDI‐MS of the isolated N‐terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALDI‐MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent structure of proteins, especially those that are refractory to standard approaches of protein chemistry.
Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NAdp+ reductase: Site‐specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials
Authors: Hurley John K. , Hazzard James T. , Martínez‐Júlvez Marta , Medina Milagros , Gómez‐Moreno Carlos , Tollin Gordon
Published: February 2009

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild‐type and seven site‐specific charge‐reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein‐protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein‐protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.
The NTR module: Domains of netrins, secreted frizzled related proteins, and type I procollagen C‐proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases
Authors: Bányai László , Patthy László
Published: February 2009

Using homology search, structure prediction, and structural characterization methods we show that the C‐terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled‐related proteins, and (4) type I procollagen C‐proteinase enhancer proteins (PCOLCEs) are homologous with the N‐terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin‐type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin‐type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.
Dissection of the protein G B1 domain binding site for human IgG Fc fragment
Authors: Sloan David J. , Hellinga Homme W.
Published: February 2009

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine‐scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well‐defined “knobs‐into‐holes” interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar “hot spot.”
Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large ΔCp's
Authors: Sheshadri S. , Lingaraju G. M. , Varadarajan R.
Published: February 2009

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8‐anilino‐1‐naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of ΔG°, ΔCp, and Cm were 3.1 plusmn; 0.2 kcal mol−1, 5.9 plusmn; 0.8 kcal mol−1 K−1 (15.9 cal (mol‐residue)−1 K−1), and 0.8 M, respectively, at pH3.0 and 14.5 plusmn; 0.4 kcal mol−1, 8.3 plusmn; 0.7 kcalmol−1 K−1 (22.4kcal (mol‐residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of ΔG° and ΔCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of ΔCp per mol‐residue for the molten globule is comparable to corresponding values of ΔCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of ΔCp per mol‐residue for the unfolding of the native state is among the highest currently known for any protein.
A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases
Authors: Makarova Kira S. , Aravind L. , Koonin Eugene V.
Published: February 2009

Computer analysis using profiles generated by the PSI‐BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'.Onthe basis of the experimentally demonstrated activity of the Methano‐bacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.
Ca2+‐dependent activity of human DNase I and its hyperactive variants
Authors: Pan Clark Q. , Lazarus Robert A.
Published: February 2009

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double‐stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300‐fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild‐type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+‐dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild‐type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26‐fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate‐enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.
Predicting allosteric switches in myosins
Authors: Kirshenbaum Kent , Young Malin , Highsmith Stefan
Published: February 2009

The sequences of several members of the myosin family of molecular motors are evaluated using ASP (Ambivalent Structure Predictor), a new computational method. ASP predicts structurally ambivalent sequence elements by analyzing the output from a secondary structure prediction algorithm. These ambivalent sequence elements form secondary structures that are hypothesized to function as switches by undergoing conformational rearrangement. For chicken skeletal muscle myosin, 13 discrete structurally ambivalent sequence elements are identified. All 13 are located in the heavy chain motor domain. When these sequence elements are mapped into the myosin tertiary structure, they form two compact regions that connect the actin binding site to the adenosine 5′‐triphosphate (ATP) site, and the ATP site to the fulcrum site for the force‐producing bending of the motor domain. These regions, predicted by the new algorithm to undergo conformational rearrangements, include the published known and putative switches of the myosin motor domain, and they form plausible allosteric connections between the three main functional sites of myosin. The sequences of several other members of the myosin I and II families are also analyzed.
Increasing protein stability by altering long‐range coulombic interactions
Authors: Grimsley Gerald R. , Shaw Kevin L. , Fee Lanette R. , Alston Roy W. , Huyghues‐Despointes BEATRICE M.P. , Thurlkill Richard L. , Scholtz J. Martin , Pace C. Nick
Published: February 2009

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI ≈︁ 3.5 and a net charge of ≈︁ –6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 Å from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild‐type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild‐type at pH 6, where the histidine 49 side chain (pKa= 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild‐type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.
Role of the prosequence of guanylin
Authors: Schulz Axel , Marx Ute C. , Hidaka Yuji , Shimonishi Yasutsugu , Rösch Paul , Forssmann Wolf‐Georg , Adermann Knut
Published: February 2009

Guanylin is a guanylyl cyclase (GC)‐activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15‐residue guanylin, representing the COOH‐terminal part of the prohormone, is released from the prohormone by cleavage of an Asp‐Pro amide bond under conditions applied during the isolation procedures. Thus, the 15‐residue guanylin is probably a non‐native, chemically induced GC‐activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15‐residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2‐terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2‐terminal region contributes significantly to the correct disulfide‐coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of α‐helical and, to a lesser extent, β‐sheet structural elements. In addition, a close proximity of the NH2‐ and the COOH‐terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH‐terminal domain from the receptor.
Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras‐proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha‐ras‐p21
Authors: Kuppens Steven , Díaz José Fernando , Engelborghs Yves
Published: February 2009

This work experimentally confirms the pathway of activation of Ha‐ras‐p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434–451). The process can be studied experimentally by analyzing the binding of BeF3− to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138–23143). Two mutants, V29G and I36G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.
Exchanging the active site between phytases for altering the functional properties of the enzyme
Authors: Lehmann Martin , Lopez‐Ulibarri Rual , Loch Claudia , Viarouge Céline , Wyss Markus , Van Loon Adolphus P.G.M.
Published: February 2009

By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase‐1, that was 15‐26 °C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase‐1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH‐activity profile. For the new protein termed consensus phytase‐7, a major—although not complete—shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 °C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase‐7 still was >7 °C more thermostable than all wild‐type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a “preferred” active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.
Two‐state vs. multistate protein unfolding studied by optical melting and hydrogen exchange
Authors: Mayne Leland , Englander S. Walter
Published: February 2009

A direct conflict between the stabilization free energy parameters of cytochrome c determined by optical methods and by hydrogen exchange (HX) is quantitatively explained when the partially folded intermediates seen by HX are taken into account. The results support the previous HX measurements of intermediate populations, show how intermediates can elude the standard melting analysis, and illustrate how they confuse the analysis when they are significantly populated within the melting transition region.
The binding of myristoylated N‐terminal nonapeptide from neuron‐specific protein CAP‐23/NAP‐22 to calmodulin does not induce the globular structure observed for the calmodulin—nonmyristoylated peptide complex
Authors: Hayashi Nobuhiro , Izumi Yoshinobu , Titani Koiti , Matsushima Norio
Published: February 2009

CAP‐23/NAP‐22, a neuron‐specific protein kinase C substrate, is Nα‐myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848‐11853) have recently found that the myristoylated N‐terminal nonapeptide of CAP‐23/NAP‐22 (mC/N9) binds to Ca2+‐bound CaM (Ca2+/CaM). In the present study, small‐angle X‐ray scattering was used to investigate structural changes of Ca2+/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca2+/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca2+/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca2+/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (Rg) for the Ca2+/CaM‐mC/N9 complex was 19.8 ± 0.3 Å. This value was significantly smaller than that of Ca2+/CaM (21.9 ± 0.3 Å), which adopted a dumbbell structure and was conversely 2‐3 Å larger than those of the complexes of Ca2+/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 Å, which was mainly due to the dumbbell structure. These results suggest that Ca2+/CaM interacts with Nα‐myristoylated CAP‐23/NAP‐22 differently than it does with other nonmyristoylated target proteins. The N‐terminal amino acid sequence alignment of CAP‐23/NAP‐22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP‐23/NAP‐22 to Ca2+/CaM, but also in the protein‐protein interactions related to other myristoylated proteins.
Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI
Authors: Goedken Eric R. , Keck James L. , Berger James M. , Marqusee Susan
Published: February 2009

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo‐form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three‐state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.
Lipoylating and biotinylating enzymes contain a homologous catalytic module
Authors: Reche Pedro A.
Published: February 2009

Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl‐ and lipoyl‐dependent enzymes is post‐translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation. Counterparts of the E. coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported. Computational analysis based on PSI‐BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module. Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X‐ray crystal structure of the E. coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes.
Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin
Authors: Galea Charles A. , Dalrymple Brian P. , Kuypers Ron , Blakeley Robert
Published: February 2009

The substrate specificity of porcine pepsin has been altered by site‐directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys‐Pro‐Xaa‐Yaa‐Phe*Nph‐Arg‐Leu (where Xaa is Ile or Pro, Yaa is Glu, Leu, Gln or Lys, Nph is p‐nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two‐ to fourfold higher kcat/Km, compared with wild‐type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild‐type pepsin cleaved a peptide corresponding to the carboxy‐terminal telopeptide region of bovine type I collagen α1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp‐Leu, Leu‐Ser, and Phe‐Leu) and at a significantly lower rate at Ser‐Phe. However, Thr222Val/Glu287Met cleaved site Ser‐Phe at a rate 20‐fold higher than the wild‐type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu‐Ser (similar to cathepsin D) and at a rate 23‐fold higher than the wild‐type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.
Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy
Authors: Bouchard Mario , Zurdo Jesús , Nettleton Ewan J. , Dobson Christopher M. , Robinson Carol V.
Published: February 2009

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature‐induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native‐like α‐helical structure. On heating to 70°C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT‐IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from α‐helical to β‐sheet structure. At later stages, EM shows the development of fibrils with well‐defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.
Comparison of binding energies of SrcSH2‐phosphotyrosyl peptides with structure‐based prediction using surface area based empirical parameterization
Authors: Henriques Denise A. , Ladbury John E. , Jackson Richard M.
Published: February 2009

The prediction of binding energies from the three‐dimensional (3D) structure of a protein–ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent‐accessible surface area‐based calculations for the prediction of the binding of Src‐SH2 domain with a series of tyrosyl phosphopeptides based on the high‐affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+3 position has been changed. Two other peptides based on the C‐terminal regulatory site of the Src protein and the platelet‐derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area‐based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (ΔCp, ΔS, ΔH, and ΔG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent‐accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters; however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.
Protein renaturation by the liquid organic salt ethylammonium nitrate
Authors: Summers Catherine A. , Flowers Robert A.
Published: February 2009

The room‐temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured‐reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one‐step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded‐active protein can be separated from the molten salt by simple desalting methods. Although the use of a low‐temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.
Purification and refolding of vascular endothelial growth factor‐B
Authors: Scrofani Sergio D.B. , Fabri Louis J. , Xu Ping , Maccarone Pino , Nash Andrew D.
Published: February 2009

Vascular endothelial growth factor (VEGF)‐A interacts with the receptor tyrosine kinases VEGF‐R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF‐A include VEGF‐B, VEGF‐C, VEGF‐D, and placenta growth factor (PLGF). Compared with VEGF‐A, relatively little is known about the biological role of the VEGF‐R1 specific ligand, VEGF‐B. Two splice variant isoforms that differ at the COOH‐terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF‐B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29‐E35). To facilitate further characterization of VEGF‐B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF‐B, including the ability to heterodimerize with endogenous VEGF‐A when co‐expressed in mammalian cells, a complex secondary structure incorporating inter‐ and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide‐linked homodimer was demonstrated to bind to VEGF‐R1 and to compete with VEGF‐A for binding to this receptor.
For the record: The three‐dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase‐NADPH‐L‐2‐amino‐6‐methylene‐pimelate
Authors: Cirilli Maurizio , Scapin Giovanna , Sutherland Andrew , Vederas John C. , Blanchard John S.
Published: February 2009

The three‐dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate d‐dehydrogenase in a ternary complex with NADPH and l‐2‐amino‐6‐methylene‐pimelate has been solved and refined to a resolution of 2.1 Å. l‐2‐Amino‐6‐methylene‐pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 μM) vs. meso‐diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+‐dependent oxidation of the d‐amino acid stereocenter of meso‐diaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a d‐amino acid. The enzyme is involved in the biosynthesis of meso‐diaminopimelate and l‐lysine from l‐aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated l‐2‐amino‐6‐methylene‐pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso‐diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the α‐l‐amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.
Primary structure of a novel subunit in ba3‐cytochrome oxidase from thermus thermophilus
Authors: Soulimane Tewfik , Buse Gerhard , Dewor Manfred , Than Manuel E. , Huber Robert
Published: February 2009

The ba3‐type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix “subunit IIa” spanning the membrane. The hydrophobic N‐terminally blocked protein was isolated in high yield using high‐performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C‐terminal sequencing of the native protein.

The molecular mass of 3,794 Da as determined by MALDI‐MS and by ESI requires the N‐terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5‐Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3‐cytochrome oxidase from Natronobacterium pharaonis.

The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).
Integrity of thermus thermophilus cytochrome c552 Synthesized by escherichia coli cells expressing the host‐specific cytochrome c maturation genes, ccmABCDEFGH: Biochemical, spectral, and structural characterization of the recombinant protein
Authors: Fee James A. , Chen Ying , Todaro Thomas R. , Bren Kara L. , Patel Kirti M. , Hill Michael G. , Gomez‐Moran Ester , Loehr Thomas M. , Ai Jingyuan , Thöny‐meyer Linda , Williams Pamela A. , Sturam Enrico , Sridhar Vandana , Mcree Duncan E.
Published: February 2009

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid‐borne, chimeric cycA gene encoding an Escherichia coli‐compatible, N‐terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid‐borne, host‐specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme‐copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H‐NMR spectroscopies. The 1.7 Å resolution X‐ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629–644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.
Structure of the Ca2+‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure
Authors: Liu Zhi‐Jie , Vysotski Eugene S. , Vysotski Eugene S. , Chen Chun‐Jung , Rose John P. , Lee John , Wang Bi‐Cheng
Published: February 2009

The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 Å resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2‐position. The protein‐coelenterazine 2‐oxy complex observed in the crystals is photo‐active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.
Fragment complementation of calbindin D28k
Authors: Berggård Tord , Thulin Eva , Linse Sara , Åkerfeldt Karin S.
Published: February 2009

Calbindin D28k is a highly conserved Ca2+‐binding protein abundant in brain and sensory neurons. The 261‐residue protein contains six EF‐hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF‐hands each (residues 1–132 and 133–261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion‐exchange and size‐exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF‐hands 1, 2, 3, or 4. The C‐terminal half (residues 133–261) appears to have a lower intrinsic stability compared to the N‐terminal half (residues 1–132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1–132 and 133–261 fragments. The affinity between these two fragments is 2 × 107 M−1, with association and dissociation rate constants of 2.7 × 104 M−1 s−1 and 1.4 × 10−3 s−1, respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual α‐helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The α‐helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+‐induced folding of the fragments is important for successful reconstitution of calbindin D28k.
Internal motional amplitudes and correlated bond rotations in an α‐helical peptide derived from 13C and 15N NMR relaxation
Authors: Idiyatullin Djaudat , Krushelnitsky Alexei , Nesmelova Irina , Daragan Vladimir A. , Mayo Kevin H. , Blanco Francesco , Serrano Luis
Published: February 2009

Peptide GFSKAELAKARAAKRGGY folds in an α‐helical conformation that is stabilized by formation of a hydrophobic staple motif and an N‐terminal capping box (Munoz V, Blanco FJ, Serrano L, 1995, Struct Biol 2:380–385). To investigate backbone and side‐chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C‐ and 15N‐enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20° and backbone ϕ,ψ bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Cα‐H, Cα‐Cβ, and N‐H rocking‐type motions along the helix dipole axis. L7 side‐chain CβH2 and CγH motions are also correlated and as motionally restricted as backbone CαH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling‐in‐a‐cone type motions dominate in water, whereas in TFE, the Cβ‐Cγ bond and phenyl ring fluctuate more simply about the Cα‐Cβ bond. These data support the Daragan‐Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378–8388) and contribute to a general understanding of internal motions in peptides and proteins.
Solution structure of DinI provides insight into its mode of RecA inactivation
Authors: Ramirez Benjamin E. , Bax Ad , Voloshin Oleg N. , Camerini‐otero R. Daniel
Published: February 2009

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81‐residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an α/β fold comprised of a three‐stranded β‐sheet and two α‐helices. The β‐sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C‐terminal α‐helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single‐stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.
The identification of conserved interactions within the SH3 domain by alignment of sequences and structures
Authors: Larson Stefan M. , Davidson Alan R. , Davidson Alan R.
Published: February 2009

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein–protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide‐binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side‐chain to main‐chain hydrogen bonds were observed in the functionally crucial RT‐Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved β‐bulge was identified in the second β‐strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.