bluuebunny/crossref_metadata_2025 huggingface dataset:
A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H‐, 13C‐NMR, and mutational analysis
Authors: Guerois Raphaël , Cordier‐Ochsenbein Françoise , Neumann Jean‐Michel , Sanson Alain , Baleux Françoise , Huynh‐Dinh Tam
Published: February 2009

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A‐helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3XXQ6 and D8XXE11. The former corresponds to the native hydrogen bond network stabilizing the α helix N‐terminus in the protein; the latter is a non‐native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D‐NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue D8. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the δ13Cα chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.
About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)
Authors: Rao K. Sudhindra , Lederer Florence
Published: February 2009

Flavocytochrome b2 or L‐lactate dehydrogenase from yeasts catalyzes the oxidation of L‐lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L‐lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate α‐hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on Nϵ of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2‐3H] lactate and the keto acid reverse substrate concentration enables the determination of the first‐order rate constant, kHe for exchange of the substrate‐derived protein‐bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115‐11122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second‐order rate constant for exchange is then 0.16 (±0.03) M−1 s−1. Using the Eigen equation, this figure yields a pKa of 9.1 ± 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291‐295). The mechanistic significance of these results is discussed.
A biomimetic strategy in the synthesis and fragmentation of cyclic protein
Authors: Tam James P. , Lu Yi‐An
Published: February 2009

This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine‐knot motif with three disulfide bonds and an end‐to‐end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol‐thiolactone exchanges to arrive at an α‐amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S, N‐acyl migration to form the cyclic protein. A two‐step disulfide formation strategy is employed for obtaining the desired disulfide‐paired products. Partial acid hydrolysis through intramolecular acyl transfer of X‐Ser, X‐Thr, Asp‐X, and Glu‐X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine‐knot motif of 1‐4, 2‐5, and 3‐6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end‐to‐end cyclic peptides and small proteins.
Porin mutants with new channel properties
Authors: Schmid Benedikt , Maveyraud Laurent , Krömer Markus , Schulz Georg E.
Published: February 2009

The general diffusion porin from Rhodopseudomonas blastica was produced in large amounts in Escherichia coli inclusion bodies and (re)natured to the exact native structure. Here, we report on 13 mutants at the pore eyelet giving rise to new diffusion properties as measured in planar lipid bilayer experiments. The crystal structures of seven of these mutants were established. The effects of charge‐modifying mutations at the pore eyelet are consistent with the known selectivity for cations. Deletions of 16 and 27 residues of the constriction loop L3 resulted in labile trimers and pores. The reduction of the eyelet cross section by introducing tryptophans gave rise to a closely correlated decrease of the conductivities. A mutant with six newly introduced tryptophans in the eyelet closed its pore in a defined manner within seconds under a voltage of 20 mV, suggesting the existence of two states. The results indicate that the pore can be engineered in a rational manner.
Structure and stability of the P93G variant of ribonuclease A
Authors: Schultz L. Wayne , Hargraves Steven R. , Klink Tony A. , Raines Ronald T.
Published: February 2009

The peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans‐to‐cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site‐directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X‐ray diffraction analysis to a resolution of 1.7 Å. This structure is essentially identical to that of the wild‐type protein, except for the 91‐94 β‐turn containing the substitution. In the wild‐type protein, the β‐turn is of type V1a. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P1 14G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of ΔΔGm, which reports on the stability lost in the variants, is 1.5‐fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91‐94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.
The discoidin domain family revisited: New members from prokaryotes and a homology‐based fold prediction
Authors: Baumgartner Stefan , Hofmann Kay , Bucher Philipp , Chiquet‐Ehrismann Ruth
Published: February 2009

Members of the discoidin (DS) domain family, which includes the C1 and C2 repeats of blood coagulation factors V and VIII, occur in a great variety of eukaryotic proteins, most of which have been implicated in cell‐adhesion or developmental processes. So far, no three‐dimensional structure of a known example of this extracellular module has been determined, limiting the usefulness of identifying a new sequence as member of this family. Here, we present results of a recent search of the protein sequence database for new DS domains using generalized profiles, a sensitive multiple alignment‐based search technique. Several previously unrecognized DS domains could be identified by this method, including the first examples from prokaryotic species. More importantly, we present statistical, structural, and functional evidence that the Dl domain of galactose oxidase whose three‐dimensional structure has been determined at 1.7 Å resolution, is a distant member of this family. Taken together, these findings significantly expand the concept of the DS domain, by extending its taxonomic range and by implying a fold prediction for all its members. The proposed alignment with the galactose oxidase sequence makes it possible to construct homology‐based three‐dimensional models for the most interesting examples, as illustrated by an accompanying paper on the C1 and C2 domains of factor V.
Identification of a frizzled‐like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases
Authors: Saldanha Jose , Singh Juswinder , Mahadevan Daruka
Published: February 2009

In Drosophila, members of the Frizzled family of tissue‐polarity genes encode proteins that appear to function as cell‐surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine‐rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine‐rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle‐specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and RORl and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt‐like ligands may bind to these developmental tyrosine kinases
An internal affinity‐tag for purification and crystallization of the siderophore receptor fhua, integral outer membrane protein from escherichia coli K‐12
Authors: Ferguson Andrew D. , Coulton James W. , Breed Jason , Diederichs Kay , Welte Wolfram
Published: February 2009

FhuA (Mr 78, 992, 714 amino acids), siderophore receptor for ferrichrome‐iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quanties sufficient for crystallization, a hexahisti‐dine tag was genetically inserted into the fhuA gene after amino acid 40.5, which resides in a known surface‐exposed loop. Recom‐binant FhuA40.5.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal‐chelate chroma‐tography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X‐ray diffraction analysis. On a rotating anode X‐ray source, diffraction was observed to 3.0 Å resolution. The crystals belong to space group P61 or P65 with unit cell dimensions of a = b = 174 Å, c = 88 Å (α = β = 90°,γ = 120°).
The interaction of eIF4E with 4E‐BP1 is an induced fit to a completely disordered protein
Authors: Fletcher C. Mark , Wagner Gerhard
Published: February 2009

4E binding protein 1 (4E‐BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4–10, 43–46, and 56–65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady‐state {1H}−15N NOEs were measured for 45 residues in the assigned regions. Except for the two C‐terminal residues, the NOEs were between −0.77 and −1.14, indicating a high level of flexibility. Furthermore, the {1H}−15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E‐BPI has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.
Detecting distant relatives of mammalian LPS‐binding and lipid transport proteins
Authors: Beamer Lesa J. , Fischer Daniel , Eisenberg David
Published: February 2009

In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three‐dimensional structure determined is bactericidal/permeability‐increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence‐sequence and sequence‐structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the “twilight zone,” their high sequence‐structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.
Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily
Authors: Thaller Maria Cristina , Schippa Serena , Rossolini Gian Maria
Published: February 2009

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol‐phosphatase domains of the bacterial bifunctional enzymes imidazole‐glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose‐6‐phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]‐D‐[ILFRMVY]‐D‐[GSNDE]‐[TV]‐[ILVAM]‐[ATS VILMC]‐X‐{YFWHKR}‐X‐{YFWHNQ}‐X( 102, 191 )‐{KRHNQ}‐G‐D‐{FYWHILVMC}‐{QNH}‐{FWYGP}‐D‐{PSNQYW}. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as “DDDD” after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.
All in the family: Structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly
Authors: Bergdoll Marc , Eltis Lindsay D. , Cameron Alexander D. , Dumas Philippe , Bolin Jeffrey T.
Published: February 2009

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modem family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal‐binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three‐dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.
Improving the binding affinity of an antibody using molecular modeling and site‐directed mutagenesis
Authors: Casipit Clayton L. , Tal Rony , Wittman Vaughan , Chavaillaz Pierre‐André , Arbuthnott Kathy , Weidanz Jon A. , Jiao Jin‐An , Wong Hing C.
Published: February 2009

Activated Factor X releases F1.2, a 271‐amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine‐amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (KD1.22 × 10−6M). To model the TA1 antibody, we entered the sequence information of the cloned TA1Fvinto the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein fromBacillus licheniformisinEscherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.
Correlation between the 1.6 Å crystal structure and mutational analysis of keratinocyte growth factor
Authors: Osslund Timothy D. , Syed Rashid , Singer Elizabeth , Hsu Eric W.‐J. , Nybo Rebecca , Harvey Timothy , Arakawa Tsutomu , Narhi Linda Owers , Chirino Arthur , Morris Charles F. , Chen Bao‐Lu
Published: February 2009

A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (A23KGF‐Rl44Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a β‐trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti‐parallel β‐strands in which three pairs of the strands form a six‐stranded beta‐barrel structure and the other three pairs of β‐strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well‐defined beta strands, which form five double‐stranded anti‐parallel beta‐sheets. A sixth poorly defined β‐strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24‐33) compared to the other FGF structures, which are important for biological activity. Based on mutapenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His 127 predominantly (onfer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of Δ23KGF‐Rl44Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main‐chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.
Binding of a substrate analog to a domain swapping protein: X‐ray structure of the complex of bovine seminal ribonuclease with uridylyl(2′,5′)adenosine
Authors: Vitagliano Luigi , Adinolfi Salvatore , Riccio Antonio , Sica Filomena , Zagari Adriana , Mazzarella Lelio
Published: February 2009

Bovine seminal ribonuclease (BS‐RNase) is a unique member of the pancreatic‐like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N‐terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2′,5′)adenosine is reported at 2.06 Å resolution. The refined model has a crystallographic R‐factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, Hisl2, Lys41, His119, and Phel20) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS‐RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.
Analysis of zinc binding sites in protein crystal structures
Authors: Alberts Ian L. , Nadassy Katalin , Wodak Shoshana J.
Published: February 2009

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H‐bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi‐zinc sites. We find that H‐bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec‐His‐Zn motif. Analysis of the stereochemistry shows that carboxyl elec‐His‐Zn motifs are geometrically rigid, while water elec‐His‐Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.
Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments
Authors: Yokota Atsushi , Takenaka Hidekazu , Oh Tomohiro , Noda Yasuo , Segawa Shin‐Ichi
Published: February 2009

Two peptide fragments from tuna cytochrome c (cyt c), N‐fragment (residues 1‐44 containing the heme) and C‐fragment (residues 45‐103), combine to form a 1:1 fragment complex. This was clearly proved by ion‐spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (ΔG°b, ΔHb, ΔS°b, and ΔCbp) associated with the complex formation were determined at various pHs and temperatures. ΔHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (ΔCbp) was directly determined from the temperature dependence of ΔHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/ dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.
Role of the 6‐20 disulfide bridge in the structure and activity of epidermal growth factor
Authors: Barnham Kevin J. , Torres Allan M. , Norton Raymond S. , Alewood Dianne , Alewood Paul F. , Domagala Teresa , Nice Edouard C.
Published: February 2009

Two synthetic analogues of murine epidermal growth factor, [Abu6, 20 mEGF4‐48 (where Abu denotes amino‐butyric acid) and [Gl, M3, K21, H40] mEGF1‐48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4‐48 was designed to determine the contribution of the 6‐20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6‐20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N‐terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main β‐sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4‐48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4‐48, implying that the structural changes in [Abu6, 20] mEGF4‐48, although limited to the N‐terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N‐terminal region with part of the receptor binding surface of EGF. [Gl, M3, K21, H40] mEGF1‐48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.
Probing enzyme quaternary structure by combinatorial mutagenesis and selection
Authors: Macbeath Gavin , Kast Peter , Hilvert Donald
Published: February 2009

Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain‐swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.
Selecting near‐native conformations in homology modeling: The role of molecular mechanics and solvation terms
Authors: Janardhan Ajit , Vajda Sandor
Published: February 2009

A free energy function, combining molecular mechanics energy with empirical solvation and entropic terms, is used for ranking near‐native conformations that occur in the conformational search steps of homology modeling, i.e., side‐chain search and loop closure calculations. Correlations between the free energy and RMS deviation from the X‐ray structure are established. It is shown that generally both molecular mechanics and solvation/entropic terms should be included in the potential. The identification of near‐native backbone conformations is accomplished primarily by the molecular mechanics term that becomes the dominant contribution to the free energy if the backbone is even slightly strained, as frequently occurs in loop closure calculations. Both terms become equally important if a sufficiently accurate backbone conformation is found. Finally, the selection of the best side‐chain positions for a fixed backbone is almost completely governed by the solvation term. The discriminatory power of the combined potential is demonstrated by evaluating the free energies of protein models submitted to the first meeting on Critical Assessment of techniques for protein Structure Prediction (CASP1), and comparing them to the free energies of the native conformations.
Aromatic L‐amino acid decarboxylase: Conformational change in the flexible region around arg334 is required during the transaldimination process
Authors: Ishii Seiji , Hayashi Hideyuki , Okamoto Akihiro , Kagamiyama Hiroyuki
Published: February 2009

Aromatic L‐amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C‐terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L‐dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine.

For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327‐Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L‐dopa as substrate, but its kcar value was decreased 57‐fold, and the Km value was increased 9‐fold compared with those of the wild‐type AADC. The absorption spectra of the fragmentary AADC in the presence of L‐dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328‐Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L‐dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.
Opposite behavior of two isozymes when refolding in the presence of non‐ionic detergents
Authors: Doñate Fernando , Artigues Antonio , Iriarte Ana , Martinez‐Carrion Marino
Published: February 2009

GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez‐Carrion M, 1995,J Biol Chem 270:1138‐1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non‐ionic detergents such as Triton X‐100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate‐limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent‐bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding β‐cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents.
Turn scanning by site‐directed mutagenesis: Application to the protein folding problem using the intestinal fatty acid binding protein
Authors: Kim Keehyuk , Frieden Carl
Published: February 2009

We have systematically mutated residues located in turns between β‐strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all β‐sheet protein exhibiting a topology comprised of two five‐stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel β‐strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C‐terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C‐terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D‐E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.
Crystallization of recombinant human heme oxygenase‐1
Authors: Schuller David J. , Poulos Thomas L. , Wilks Angela , Montellano Paul Ortiz De
Published: February 2009

Heme oxygenase catalyzes the NADPH, 02, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO‐1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C‐terminus. While full‐length human HO‐1 consists of 288 residues, a truncated version with residues 1‐265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N‐terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1‐226 and 1‐237. Expression of the 1‐226 and 1‐233 versions of human HO‐1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P21, with unit cell dimensions a = 79.3 Å, b = 56.3 Å, c = 112.8 Å, and β = 101.5°.
Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories
Authors: Taylor Marc G. , Kirsch Jack F. , Rajpal Arvind
Published: February 2009

The rate constants, kon, for the formation of hen (chicken) lysozyme (HEWL) Fab‐ l0 complexes have been determined for wild‐type (WT) and epitope‐mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10‐fold range [(0.18 to 1.92) × 106 M−1 s−1]. The affinity constants, KD, cover a broader, 440‐fold, range from 0.075 to 33 nM. Values of KD as high as 7 μM were obtained for the complexes prepared from some mutations at HEWL positions 96 and 97, but the associated kinetic constants could not be determined. The values of kon are negatively correlated with side‐chain volume at position 101HEWL, but are essentially independent of this parameter for position 21hewl substitutions. The multiple mutations made at positions 21HEWL and 101HEWL provide sufficient experimental data on complex formation to evaluate Φ values [Φ = (ΔΔG‡on)/(ΔΔGD)] at these two positions to begin to define trajectories for protein‐protein association. The data, when interpreted within the concept of a two‐step association sequence embracing a metastable encounter complex intermediate, argue that the rate determining step at position 2lHEWL (Φavg = 0.2) is encounter complex formation, but the larger Φavg value of 0.36 experienced for most position 10lHEWL mutations indicates a larger contribution from the post‐encounter annealing process at this site for these replacements.
Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design
Authors: Liang Jie , Woodward Clare , Edelsbrunner Herbert
Published: February 2009

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure‐based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme‐ligand structures, several elastase‐inhibitor complexes, the FK506 binding protein, 30 HIV‐1 protease‐inhibitor complexes, and a number of small and large protein inhibitors. Medium‐sized globular proteins typically have 10‐20 pockets/cavities. Most often, binding sites are pockets with 1‐2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 102‐103Å3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < 700 Å3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994,Nat Struct Bid 1:55‐58). Analysis of elastase‐inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996,Science274:1531‐1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked‐fragment design. CAST analysis of 30 HIV‐1 protease‐inhibitor complexes shows that the flexible active site pocket can vary over a range of 853‐1,566 Å3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.
Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: Plant‐specific mononuclear blue copper proteins
Authors: Nersissian Aram M. , Valentine Joan Selverstone , Immoos Chad , Hill Michael G. , Hart P. John , Williams Gregory , Herrmann Reinhold G.
Published: February 2009

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arubidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant‐specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arubidopsis. while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper‐binding amino acids. The recombinant copper binding domain of Arubidopsis uclacyanin I was expressed, purified, and shown to bind a copper atom in a fashion known as “blue” or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin I rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild‐type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron‐transfer proteins participating in long‐range electron‐transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.
Lysine‐50 is a likely site for anchoring the plasminogen N‐terminal peptide to lysine‐binding kringles
Authors: An Seong Soo A. , Marti Daniel N. , Llinas Miguel , Carreño Cristina , Albericio Fernando , Schaller Johann
Published: February 2009

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N‐terminal Glul‐Lys77 peptide (NTP) have been investigated via 1H‐NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM−1). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nα‐acetyl‐L‐lysine‐methyl ester (Ka ˜ 0.2 mM−1), peptides encompassing Lys50 consistently show higher Ka values. Among the investigated linear peptides, Nα‐acetyl‐Ala‐Phe‐Tyr‐His‐Ser‐Ser‐Lys50‐Glu‐Gln‐NH2 (AcAFYHSKSOEQ‐NH2) exhibits the strongest interaction with K4 (Ka ˜ 1.4 mM−1), followed by AcYHSKSOEQ‐NH2 (Ka ˜ 0.9 mM−1). Relative to the wild‐type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 → Ser mutation was introduced (⇒ AcYHSSSOEQ‐NH2), binding was abolished.

The Ile27‐Ile56 construct (L‐NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rKI) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L‐NTP, was investigated. We find that L‐NTP interacts with rK1, rK2, K4, and K5 — all lysine‐binding kringles — in a fashion that closely mimics what has been observed for the Glul‐HSer57 N‐terminal fragment of Pgn (CB‐NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L‐NTP and magnitudes of kringle‐L‐NTP binding affinities (rK1, K2 ˜ 4.3 mM −1; rK2, Ka ˜ 3.7 mM−1; K4, Ka ˜ 6.4 mM−1; and K5, Ka ˜ 2.1 mM−1) are essentially the same as for the corresponding kringle‐CB‐NTP pairs. Molecular modeling studies suggest that the GIu39‐Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent‐exposed kringle LBS surface.