bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease
Authors: Fink Anthony L. , Calciano Linda J. , Goto Yuji , Nishimura Midori , Swedberg Sally A.
Published: February 2009

Titration of a salt‐free solution of native staphylococcal nuclease by HC1 leads to an unfolding transition in the vicinity of pH 4, as determined by near‐ and far‐UV circular dichroism. At pH 2‐3, the protein is substantially unfolded. The addition of further HC1 results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1‐anilino‐8‐naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid‐unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in α‐helix structure, with a corresponding increase in β or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.
Refolding and proton pumping activity of a polyethylene glycol‐bacteriorhodopsin water‐soluble conjugate
Authors: Sirokmán Géza , Fasman Gerald D.
Published: February 2009

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m‐PEG; molecular weight = 5, 000 Da) succinimidyl carbonate. The polyethylene glycol‐bacteriorhodopsin (m‐PEG‐SC‐BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain α‐helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent‐lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m‐PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m‐PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m‐PEG‐SC‐BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.
Cross‐validation of protein structural class prediction using statistical clustering and neural networks
Authors: Metfessel Brent A. , Connelly Donald P. , Rich Stephen S. , Saurugger Peter N.
Published: February 2009

We present an approach to predicting protein structural class that uses amino acid composition and hydrophobic pattern frequency information as input to two types of neural networks: (1) a three‐layer back‐propagation network and (2) a learning vector quantization network. The results of these methods are compared to those obtained from a modified Euclidean statistical clustering algorithm. The protein sequence data used to drive these algorithms consist of the normalized frequency of up to 20 amino acid types and six hydrophobic amino acid patterns. From these frequency values the structural class predictions for each protein (all‐alpha, all‐beta, or alpha‐beta classes) are derived. Examples consisting of 64 previously classified proteins were randomly divided into multiple training (56 proteins) and test (8 proteins) sets. The best performing algorithm on the test sets was the learning vector quantization network using 17 inputs, obtaining a prediction accuracy of 80.2%. The Matthews correlation coefficients are statistically significant for all algorithms and all structural classes. The differences between algorithms are in general not statistically significant. These results show that information exists in protein primary sequences that is easily obtainable and useful for the prediction of protein structural class by neural networks as well as by standard statistical clustering algorithms.
Synthetic chimeras of mouse growth factor‐associated glandular kallikreins. I. Kinetic properties
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph A. , Burnier John P. , Marsters James C.
Published: February 2009

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor‐associated mouse glandular kallikreins–epidermal growth factor binding protein (EGF‐BP) and the γ‐subunit of nerve growth factor (γ‐NGF)–were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and β‐NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237–282, 1983), in γ‐NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the β‐NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF‐BP and γ‐NGF. Finally, in γ‐NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF‐BP and γ‐NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.
Synthetic chimeras of mouse growth factor‐associated glandular kallikreins. II. Growth factor binding properties
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph A. , Holden Hazel M.
Published: February 2009

Six chimeric constructs of the sequentially similar growth factor‐associated kallikreins–epidermal growth factor binding protein (EGF‐BP) and the γ‐subunit of nerve growth factor (γ‐NGF)–have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and β‐NGF, analogous to the high molecular weight forms (7S NGF and HMW‐EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native β‐NGF (along with native α‐NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an α2βγ2 structure (where β‐NGF is itself a dimer), with recombinant (r) γ‐NGF and with a chimera in which the amino terminal region from EGF‐BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of γ‐NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from γ‐NGF retained at least some ability to form the 7S complex. rEGF‐BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF‐BP and the carboxyl terminal segment of γ‐NGF formed a nativelike HMW‐EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and β‐NGF, respectively.
Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics
Authors: Labean Thomas H. , Kauffman Stuart A.
Published: February 2009

Libraries of random sequence polypeptides are useful as sources of unevolved proteins, novel ligands, and potential lead compounds for the development of vaccines and therapeutics. The expression of small random peptides has been achieved previously using DNA synthesized with equimolar mixtures of nucleotides. For many potential uses of random polypeptide libraries, concerns such as avoiding termination codons and matching target amino acid compositions make more complex designs necessary. In this study, three mixtures of nucleotides, corresponding to the three positions in the codon, were designed such that semirandom DNA synthesized by repeated cycles of the three mixtures created an open reading frame encoding random sequence polypeptides with desired ensemble characteristics. Two methods were used to design the nucleotide mixtures: the manual use of a spreadsheet and a refining grid search algorithm. Using design targets of less than or equal to 1% stop codons and an amino acid composition based on the average ratios observed in natural, globular proteins, the search methods yielded similar nucleotide ratios. Semirandom DNA, synthesized with a designed, three‐residue repeat pattern, can encode libraries of very high diversity and represents an important tool for the construction of random polypeptide libraries.
Independence of metal binding between tandem Cys2His2 zinc finger domains
Authors: Krizek Beth Allyn , Berg Jeremy M. , Zawadzke Laura E.
Published: February 2009

Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.
Functional consequences of mutations at the allosteric interface in hetero‐ and homo‐hemoglobin tetramers
Authors: Baudin V. , Pagnier J. , Kiger L. , Kister J. , Bihoreau M. T. , Lacaze N. , Marden M. C. , Poyart C. , Schaad O. , Edelstein S. J.
Published: February 2009

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While α chains associate weakly into dimers, β chains self‐associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the β4 tetramer is frozen in a quaternary R‐state resembling that of liganded HbA. X‐ray diffraction studies of the liganded β4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the “allosteric” interface (α1β2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T‐state of the β4 tetramer. We have studied natural and artificial Hb mutants at different sites in the β chains responsible for the T‐state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme‐heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero‐ and homotetramers. Our conclusion is that the “allosteric” interface is so precisely tailored for maintaining the assembly between αβ dimers that any change in the side chains of β40 (C6), β99 (G1), and β101 (G3) involved in the interface results in increased R‐state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the β4 hemoglobin and the formation of lower affinity noncooperative monomers.
Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis
Authors: Hess Daniel , Winz Robert , Brownsey Roger W. , Aebersold Ruedi , Covey Tom C.
Published: February 2009

We report the use of microbore reverse‐phase high performance liquid chromatography connected on‐line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow‐splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer.

The method was used to analyze the 265‐kDa and the 280‐kDa isoforms of the enzyme acetyl‐CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error‐free data interpretation in chemical high‐sensitivity peptide sequence analysis.
Hematopoietic cytokines: Similarities and differences in the structures, with implications for receptor binding
Authors: Wlodawer Alexander , Pavlovsky Alexander , Gustchina Alla
Published: February 2009

Crystal and NMR structures of helical cytokines–interleukin‐4 (IL‐4), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and interleukin‐2 (IL‐2)–have been compared. Root mean square deviations in the Cα coordinates for the conserved regions of the helices were 1–2 Å between different cytokines, about twice the differences observed for independently determined crystal and solution structures of IL‐4. Considerable similarity in amino acid sequence in the areas expected to interact with the receptors was detected, and the available mutagenesis data for these cytokines were correlated with structure conservation. Models of cytokine‐receptor interactions were postulated for IL‐4 based on its structure as well as on the published structure of human growth hormone interacting with its receptors (de Vos, A.M., Ultsch, M., & Kossiakoff, A.A., 1992, Science 255, 306–312). Patches of positively charged residues on the surfaces of helices C and D of IL‐4 may be responsible for the interactions with the negatively charged residues found in the complementary parts of the IL‐4 receptors.
Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions
Authors: Lin Xinli , Loy Jeffrey A. , Sussman Fredy , Tang Jordan
Published: February 2009

Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two‐chain pepsin or a two‐chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257–17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline‐inactivated pepsin was regenerated by the addition of the recombinant N‐terminal lobe but not by the C‐terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N‐terminal lobe. A complex between recombinant N‐terminal lobe of pepsinogen and alkaline‐denatured pepsin has been isolated. This complex is structurally similar to a two‐chain pepsinogen, but it contains an extension of a denatured pepsin N‐terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N‐terminal lobe. The structural components that are responsible for the alkaline instability of the N‐terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N‐terminal domain (as compared to 19 in the C‐terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118. Especially, the partially buried Asp11, which interacts with Asp159, could cause one of these two groups to have an abnormally high pKa and the other an abnormally low pKa value. Thus, the ionization of Asp11 at a high pH may place two negatively charged residues in close vicinity. This unfavorable situation may be the trigger for the denaturation of the N‐terminal lobe of pepsin.
The structure and function of omega loop A replacements in cytochrome c
Authors: Murphy Michael E. P. , Brayer Gary D. , Fetrow Jacquelyn S. , Burton Randall E.
Published: February 2009

The structural and functional consequences of replacing Ω‐loop A (residues 18–32) in yeast iso‐1‐cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three‐dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X‐ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild‐type yeast iso‐1‐cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of Ω‐loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, Ω‐loop A contains a valine at position 20, which restores the original wild‐type packing arrangement of the hydrophobic core. Also, as a result of Ω‐loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main‐chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild‐type yeast iso‐1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 °C. This is 19 mV less than the wild‐type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of Ω‐loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.
Interactions and inhibition of blood coagulation factor Va involving residues 311–325 of activated protein C
Authors: Mesters Rolf M. , Heeb Mary J. , Griffin John H.
Published: February 2009

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide‐(311–325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa‐1‐stage coagulation assays in normal and in protein S‐depleted plasma with 50% inhibition at 13 μM peptide. In a system using purified clotting factors, peptide‐(311–325) inhibited APC‐catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 μM peptide. However, peptide‐(311–325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]α1‐antitrypsin. Peptide‐(311–325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 μM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose‐dependent manner to immobilized peptide‐(311–325). Peptide‐(311–315) inhibited the binding of factor Va to immobilized APC or factor Xa. These data are consistent with the hypothesis that residues 311–325 in APC bind to factor Va at a site that can bind either APC or factor Xa, and that peptide‐(311–325) interferes with both APC inactivation of factor Va and expression of factor Xa activity in the prothrombinase complex by binding to this site.
Prolyl isomerases catalyze antibody folding in vitro
Authors: Lilie Hauke , Buchner Johannes , Lang Kurt , Rudolph Rainer
Published: February 2009

Some slow‐folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl‐peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate‐limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate‐limiting isomerization of Xaa‐Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction. (2) PPI and BSA are able to increase the yield of refolding of Fab by reducing the formation of aggregates or the adsorption to the surface of the reaction vessel in an unspecific manner. This behavior is clearly distinct from the mechanism of action observed with the chaperone GroEL.
Thermodynamics of apocytochrome b5 unfolding
Authors: Pfeil Wolfgang
Published: February 2009

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two‐state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (ΔG) amounts to ΔG = 7 ± 1 kJ/mol at 25 °C (pH 7.4) compared with ΔG = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, nativelike protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1–35 and 79–90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199–207).
Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations
Authors: Zheng Jianhua , Knighton Daniel R. , Taylor Susan S. , Xuong Nguyen‐Huu , Sowadski Janusz M. , Eyck Lynn F. Ten
Published: February 2009

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP‐dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154–2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 Å resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C‐terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active‐site region surrounding the glycine‐rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N‐terminal 40 residues that lie outside the conserved catalytic core are anchored by the N‐terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.
Identification, classification, and analysis of beta‐bulges in proteins
Authors: Chan A. W. Edith , Hutchinson E. Gail , Harris Daniel , Thornton Janet M.
Published: February 2009

A β‐bulge is a region of irregularity in a β‐sheet involving two β‐strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in β‐sheets were identified and classified automatically, extending the definition of β‐bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574–2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of β‐bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, β‐bulges produce two main changes in the structure of a β‐sheet: (1) disrupt the normal alternation of side‐chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands.
Solution structure of the calcium channel antagonist ω‐conotoxin GVIA
Authors: Skalicky Jack J. , Ciesla D. Joseph , Pardi Arthur , Metzler William J. , Galdes Alphonse
Published: February 2009

The three‐dimensional solution structure is reported for ω‐conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen‐bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple‐stranded β‐sheet containing a β‐hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.
Effect of a single aspartate on helix stability at different positions in a neutral alanine‐based peptide
Authors: Huyghues‐Despointes Beatrice M. P. , Scholtz J. Martin , Baldwin Robert L.
Published: February 2009

A single aspartate residue has been placed at various positions in individual peptides for which the alanine‐based reference peptide is electrically neutral, and the helix contents of the peptides have been measured by circular dichroism. The dependence of peptide helix content on aspartate position has been used to determine the helix propensity (s‐value). Both the charged (Asp−) and uncharged (Asp0) forms of the aspartate residue are strong helix breakers and have identical s‐values of 0.29 at 0°C. The interaction of Asp− with the helix dipole affects helix stability at positions throughout the helix, not only near the N‐terminus, where the interaction is helix stabilizing, and the C‐terminus, where it is destabilizing. Comparison of the helix contents at acidic pH (Asp0) and at neutral pH (Asp−) shows that the charge–helix dipole interaction is screened slowly with increasing NaCl concentration, and screening is not complete even at 4.8 M NaCl. Lastly, a helix‐stabilizing hydrogen‐bond interaction between glutamine and aspartate (spacing i, i + 4) has been found. This side‐chain interaction is specific for both the orientation and spacing of the glutamine and aspartate residues and is resistant to screening by NaCl.
Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms
Authors: Priestle John P. , Rahuel Joseph , Rink Hans , Tones Michael , Grütter Markus G.
Published: February 2009

The three‐dimensional structures of d‐Phe‐Pro‐Arg‐chloromethyl ketone‐inhibited thrombin in complex with Tyr‐63‐sulfated hirudin55–65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor d‐Phe‐Pro‐Arg‐Pro‐(Gly)4‐hirudin54–65 (CGP 50, 856, binary complex) have been determined by X‐ray crystallography in crystal forms different from those described by Skrzypczak‐Jankun et al. (Skrzypczak‐Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379–1393). In both complexes, the interactions of the C‐terminal hirudin segments of the inhibitors binding to the fibrinogen‐binding exosite of thrombin are clearly established, including residues 60–64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr‐63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10‐fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146–150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C‐terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor–thrombin complex, the peptide bond between Arg‐Pro (P1‐P1′) seems to be cleaved.
Prediction of protein conformation on the basis of a search for compact structures: Test on avian pancreatic polypeptide
Authors: Liwo A. , Wawak R. J. , Scheraga H. A. , Pincus M. R. , Rackovsky S.
Published: February 2009

Based on the concept that hydrophobic interactions cause a polypeptide chain to adopt a compact structure, a method is proposed to predict the structure of a protein. The procedure is carried out in four stages: (1) use of a virtual‐bond united‐residue approximation with the side chains represented by spheres to search conformational space extensively using specially designed interactions to lead to a collapsed structure, (2) conversion of the lowest‐energy virtual‐bond united‐residue chain to one with a real polypeptide backbone, with optimization of the hydrogen‐bond network among the backbone groups, (3) perturbation of the latter structure by the electrostatically driven Monte Carlo (EDMC) procedure, and (4) conversion of the spherical representation of the side chains to real groups and perturbation of the whole molecule by the EDMC procedure using the empirical conformational energy program for peptides (ECEPP/2) energy function plus hydration. Application of this procedure to the 36‐residue avian pancreatic polypeptide led to a structure that resembled the one determined by X‐ray crystallography; it had an α‐helix starting at residue 13, with the N‐terminal portion of the chain in an extended conformation packed against the α‐helix. Similar structures with slightly higher energies, but looser packing, were also obtained.
Echistatin disulfide bridges: Selective reduction and linkage assignment
Authors: Gray William R.
Published: February 2009

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris‐(2‐carboxyethyl)‐phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 °C a single bridge linking Cys 2‐Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 °C the bridges Cys 7‐Cys 32 and Cys 8‐Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 °C to a peptide that retains a single bridge linking Cys 20‐Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 °C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2–11; 7–32; 8–37; 20–39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.
Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models
Authors: Mann Craig J. , Royer Catherine A. , Matthews C. Robert
Published: February 2009

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild‐type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844–1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild‐type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011–7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591–597) provides a simple explanation for this phenomenon.
Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels
Authors: Grove Anne , Tomich John M. , Iwamoto Takeo , Montal Mauricio
Published: February 2009

To identify sequence‐specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel‐forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage‐gated calcium channel. The amino acid sequence of voltage‐gated, dihydropyridine (DHP)‐sensitive calcium channels suggests the presence in each of four homologous repeats (I–IV) of six segments (S1–S6) predicted to form membrane‐spanning, α‐helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four‐helix bundle motif, four‐helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation‐selective channels, but with distinct characteristics: the single‐channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by μM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.
Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry
Authors: Tsarbopoulos Anthony , Pramanik Birendra N. , Labdon James E. , Reichert Paul , Gitlin Gerry , Patel Suman , Sardana Vinod , Nagabhushan Tattanahalli L. , Trotta Paul P.
Published: February 2009

A trypsin‐resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) was isolated and analyzed by high‐energy Cs+ liquid secondary‐ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high‐mass disulfide‐linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM‐CSF provided rapid confirmation of the cDNA‐derived sequence and determination of the existing disulfide bonds between cysteine residues 54–96 and 88–121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with β‐mercaptoethanol.
Heme biosynthesis in mammalian systems: Evidence of a schiff base linkage between the pyridoxal 5′‐phosphate cofactor and a lysine residue in 5‐aminolevulinate synthase
Authors: Ferreira Gloria C. , Neame Peter J. , Dailey Harry A.
Published: February 2009

5‐Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5‐aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584–590). 5‐Aminolevulinate synthase is a pyridoxal 5′‐phosphate‐dependent enzyme and is functional as a homodimer.

The recombinant 5‐aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5′‐phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA‐derived 5‐aminolevulinate synthase sequences and is present in the C‐terminal (catalytic) domain.

Mutagenesis of the 5‐aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5′‐phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.
Amide proton exchange rates of oxidized and reduced saccharomyces cerevisiae iso‐1‐cytochrome c
Authors: Marmorino Jennifer L. , Auld Douglas S. , Betz Stephen F. , Doyle Donald F. , Young Gregory B. , Pielak Gary J.
Published: February 2009

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso‐1‐cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent‐accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in Ω‐loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent‐accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, ΔGd, at 298 K to the free energy change for local unfolding, ΔGop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in ΔGop. Comparisons of ΔGd to ΔGop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high‐energy conformations of the reduced form appears to involve the axially coordinated heme.
Application of molecular dynamics and free energy perturbation methods to metalloporphyrin‐ligand systems ii: Co and dioxygen binding to myoglobin
Authors: Lopez Marco A. , Kollman Peter A.
Published: February 2009

The protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two‐step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2–3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe‐C‐O bond is found to be bent (θ = 159.8°) with a small tilt (ϕ = 6.2°). The contributions made by each of the 29 residues — within the 9.0‐Å radius of the iron atom — to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2 → MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.
Thermodynamics of bpti folding
Authors: Makhatadze George I. , Kim Key‐Sun , Woodward Clare , Privalov Peter L.
Published: February 2009

A calorimetric study of the basic pancreatic trypsin inhibitor (BPTI) has been performed using the new generation of the adiabatic scanning microcalorimeters, operating in an extended temperature range of 5–130 °C. Precise measurements of the heat capacities of the native and unfolded states of BPTI show that the heat capacity change upon unfolding strongly depends on temperature; its value is maximal at about 50 °C and diminishes as the temperature is increased. The temperature dependencies of the enthalpy and entropy changes upon BPTI unfolding were found to be similar to those normally observed for other small globular proteins. The stability of BPTI has been correlated with its structure.
Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation
Authors: Swint Liskin , Robertson Andrew D.
Published: February 2009

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wavelengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 + 0.4 °C and the enthalpy of unfolding at this temperature (ΔHm) is 40.8 + 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of ΔHm versus Tm yield a mean value of 590 + 120 cal/(mol·K) for the change in heat capacity upon unfolding (ΔCp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 + 110 cal/(mol·K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining ΔCp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520–2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (ΔGu) were plotted versus temperature, the resulting curve appeared linear; therefore, ΔCp could not be determined. However, the data for ΔGu versus denaturant concentration are linear over an extra ordinarily wide range of concentrations. Moreover, extrapolated values of ΔGu in urea are identical to values measured directly.
Structure and orientation of the antibiotic peptide magainin in membranes by solid‐state nuclear magnetic resonance spectroscopy
Authors: Bechinger Burkhard , Zasloff Michael , Opella Stanley J.
Published: February 2009

Magainin 2 is a 23‐residue peptide that forms an amphipathic α‐helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One‐ and two‐dimensional solid‐state 15N NMR spectra of specifically 15N‐labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is α‐helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.