bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing
Authors: Boisset Nicolas , Frank Joachim , Taveau Jean Christophe , Billiald Philippe , Motta Genevieve , Lamy Josette , Sizaret Pierre Yves , Lamy Jean
Published: May 2005

Three epitopes have been localized by immunoelectron microscopy on subunits Aa6 of the 4 × 6‐meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single‐layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6: 187‐194, 1981). A high‐precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.
Lecithin: Cholesterol acyltransferase activation by synthetic amphipathic peptides
Authors: Subbarao Nanda K. , Fielding Christopher J. , Hamilton Robert L. , Szoka Francis C.
Published: May 2005

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.: 38: 247–253, 1974) proposes that the lipid‐binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30‐residue peptide, GALA, which in the alpha helical conformation hs a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26: 2964–2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphospatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC‐GALA:19/1 (molar ratio) complex can be isolated by gel‐permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44‐Å diameter. GALA edge thickness and a 170‐ to 350‐Å diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100‐fold larger than into egg phosphatidlylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1‐like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.
Preliminary X‐ray diffraction studies and biochemical characterization of the antitumor protein mitomalcin indicate close similarity to neocarzinostatin
Authors: Sieker L. C. , Gnanarajah G. G.
Published: May 2005

The antitumor antibiotic protein mitomalcin, from the microorganism Streptomyces malayensis, has been purified to apparent homogenity and crystallized. The crystals belong to space group P212121 and have the following cell parameters : a=27.2 Å, b=34.1 Å, c=101.7 Å, and alpha;=β=γ=90°. These crystal properties are extremely similar to crystals of the antitumor protein neocarzinostatin (11.7 kilodaltons [kDa]) from Streptomyces carzinostaticus in spite of differing pH conditions for crystallizing the two proteins and an apparent difference in molecular weight is similar to that of neocarzinostatin. An amino acid composition analysis of mitomalcin indicates that some differences may exist between the two molecules, but a preliminary amino acid sequence analysis of the first 37 residues found no difference in the N‐terminal region of the molecule.
Microfolding: Conformational probability map for the alanine dipeptide in water from molecular dynamics simulations
Authors: Anderson Amil G. , Hermans Jan
Published: May 2005

A direct attack on the protein‐folding problem has been initiated with the free energy perturbation methods of molecular dynamics. The complete conformational probability map for the alanine dipeptide is presented. This work uses the SPC model for the explicit hydration of the dipeptide. Free energy differences for the four observed minima (β, αR, αL, C) are given, and the free energy barriers between minima are outlined.
Criteria that discriminate between native proteins and incorrectly folded models
Authors: Novotný Jiři , Rashin A. A. , Bruccoleri R. E.
Published: May 2005

Various theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent‐exposed surface, and surface charge distribution between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the α‐helical hemerythrin were modeled on the β‐sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on he hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work,1where incorrect side chains were modeled inall transconformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s differences from the x‐ray side chains 2.2–2.4 Å were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula‐based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little criteria clearly favored the native structures over the misfolded ones: (1) solvent‐exposed side‐chain nonpolar surface, (2) number of buried ionizable groups, and (3) empirical free energy functions that incorporate solvent effects.
Structure‐function relationships in 3‐phosphoglycerate kinase: Role of the carboxy‐terminal peptide
Authors: Mas Maria T. , Resplandor Zenaida E.
Published: May 2005

Yeast 3‐phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr ∼ 45,000) composed of two globular domains. Each domain corresponds approximately to the amino‐ and carboxyterminal halves of the polypeptide chain. The carboxy‐terminal end extends over the interdomain “hinge” region and packs against the amino‐terminal domain. It has been proposed that domain movement, resulting in closure of the active site left, is essential for the catalytic of PGK. Large‐scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site‐specific mutagenesis, we have removed a 15‐amino‐acid carboxy‐terminal fragment, in order to probe its role in the substrate‐ and sulfate‐induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfateinduced activation. The Km for ATP was essentially unchanged (Km = 0.23mM), whereas the Km value for 3‐phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy‐terminal segment is important for the mechanism of substrate‐ and specific‐induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy‐terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy‐terminal peptide also contributes to the stability of PGK.
Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation
Authors: Hendrickson Wayne A. , Smith Janet L. , Phizackerley R. Paul , Merritt Ethan A.
Published: May 2005

The molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least‐squares procedure for analyzing the MAD data. MAD phases for the structure at 3Å resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.
Comparison of the dynamics of the membrane‐bound form of fd coat protein in micelles and in bilayers by solution and solid‐state nitrogen‐15 nuclear magnetic resonance spectroscopy
Authors: Bogusky M. J. , Leo G. C. , Opella S. J.
Published: May 2005

Solid‐state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane‐bound form of the protein. The residues in the N‐ and C‐terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C‐terminal region of the coat protein in micelles; at 25°C only the last two residues are mobile on the 109‐Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C‐terminal residues are immobile in the virus particles, the mobility of these residues in the membrane‐bound form of the protein may be important for the formation of protein‐DNA interactions in the assembly process.
Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison
Authors: Brünger Axel T. , Karplus Martin
Published: May 2005

A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X‐ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.
Helix lap‐joints as ion‐binding sites: DNA‐binding motifs and Ca‐binding “EF hands” are related by charge and sequence reversal
Authors: Richardson Jane S. , Richardson David C.
Published: May 2005

The DNA‐binding helix pairs in gene repressor and activator were compared with other approximately perpendicular pairs of adjacent helices in the known protein structures. Two other examples of closely matching conformations were found in cytochrome c peroxidase (residues 153–174) and in ribosomal L7/L12 protein (residues 68–89). Another group of such offset “lap‐joints” are the Ca‐binding “EF hand” structures, which bind a positive rather than a negative ligand. The EF hands turn out to match the DNA‐binding motifs quite well (outside of the loop) if their sequence direction is reversed. This conformation is thus not as unusual as had been thought, but may have a more generalized role in ion binding and occasionally occur in a purely structural role.
Test of circular dichroism (CD) methods for crambin and CD‐assisted secondary structure prediction of its homologous toxins
Authors: Teeter M. M. , Whitlow Marc
Published: May 2005

Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high‐resolution crystal structure. In addition, a two‐step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin.

The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945 Å resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107–113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S. W., Glöckner, J. Biochemistry 20:33–37, 1981) as modified (Manavalan, P., Johnson, W. C., Jr. Anal. Biochem. 167:76–85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane‐active toxins homologous to crambin (α1‐ and β‐purothionin, phoratoxin A and B, an viscotoxin A3 and B).

The new program SEQ (pronounced “seek”) was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location.

Both CD‐arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of Purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.
The construction of new proteins: V. A template‐assembled synthetic protein (TASP) containing both a 4‐helix bundle and β‐barrel‐like structure
Authors: Mutter Manfred , Hersperger René , Gubernator Klaus , Müller Klaus
Published: May 2005

The construction of a template‐assembled synthetic protein (TASP) designed to contain both a 4‐helix bundle and a β‐barrel as two folding “domains” is described. For the de novo design of proteins, amphiphilic helices (α) and β‐sheets (β) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intarmolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8‐(4α)(4β). The design of this new macromolecule was assisted by computer modeling, which suggested a low‐energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid‐phase synthesis of the “two‐domain” TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.
Electrostatic interactions in the assembly of Escherichia coli aspartate transcarbamylase
Authors: Glackin M. P. , McCarthy M. P. , Mallikarachchi D. , Matthew J. B. , Allewell N. M.
Published: May 2005

Although ionizable groups are known to play important roles in the assembly, catalytic, and regulatory mechanisms of Escherichia coli aspartate transcarbamylase, these groups have not been characterized in detail. We report the application of static accessibility modified Tanford‐Kirkwood theory to model electrostatic effects associated with the assembly of pair of chains, subunits, and the holoenzyme. All of the interchain interfaces except R1–R6 are stabilized by electrostatic interactions by −2 to −4 kcal−m−1 at pH 8. The pH dependence of the electrostatic component of the free energy of stabilization of intrasubunit contacts (C1–C2 and R1–R6) is qualitatively different from that of intersubunit contacts (C1–C4, C1–R1, and C1–R4). This difference may allow the transmission of information across subunit interfaces to be selectively regulated. Groups whose calculated pK or charge changes as a result of protein‐protein interactions have been identified and the results correlated with available information about their function. Both the 240s loop of the c chain and the region near the Zn(II) ion of the r chain contain clusters of ionizable groups whose calculated pK values change by relatively large amounts upon assembly. These pK changes in turn extend to regions of the protein remote from the interface. The possibility that networks of ionizable groups are involved in transmitting information between binding sites is suggested.
Structural basis of hierarchical multiple substates of a protein. I: Introduction
Authors: Noguti Tosiyuki , Gō Nobuhiro
Published: May 2005

A computer experiment of protein dynamics is carried out, which consists of two steps: (1) A Monte Carlo simulation of thermal fluctuations in the native state of a globular protein, bovinepancreatic trypsin inhibitor; and (2) a simulation of the quick freezingof fluctuating conformations into energy minima by minimization of the energy of a number of conformations sampled in the Monte Carlo simulations sampled in the Monte Carlo simulation. From the analysis of results of the computer experiment is obtained the following picture of protein dynamics:multiple energy minima exist in the native state, and they are distributedin clusters in the conformational space. The dynamics has a hierarchical structure which has at least two levels. In the first level, dynamics is restricted within one of the clusters of minima. In the second, transitions occur among the clusters. Local parts of a protein molecule, side chains and local main chain segments, can take multiple locally stable conformations in the native state. Many minima result from combinations of these multiple local conformations. The hierarchical structure in the dynamics comes from interactions among the local parts. Protein moleculeshave two types of flexibility, each associated with elastic and plastic deformations, respectively.
Structural basis of hierarchical multiple substates of a protein. II: Monte carlo simulation of native thermal fluctuations and energy minimization
Authors: Noguti Tosiyuki , Gō Nobuhiro
Published: May 2005

Conformational fluctuations in a globular protein, bovine pancreatic trypsin inhibitor, in the time range between picoseconds and nanoseconds are studied by a Monte Carlo simulation method. Multipleenergy minima are derived from sampled conformations by minimizing their energy. They are distributed in clusters in the conformational space. A hierarchical structure is observed in the simulated dynamics. In the time range between 10−14 and 10−10 seconds dynamics is well represented by a superposition of vibrational motions within an energy well with transitions among minima within each cluster. Transitions among clusters take place in the time range of nanoseconds or longer.
The pro‐peptide is not necessary for active renin secretion from transfected mammalian cells
Authors: Harrison T. M. , Chidgey M. A. J , Brammar W. J , Adams G. J
Published: May 2005

Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cellsto secrete the enzymically inactive pro‐protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro‐peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro‐peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.
Single crystals of bacteriophage T7 RNA polymerase
Authors: Sousa Rui , Rose John P. , Je Chung Yong , Lafer Eileen M. , Wang Bi‐Cheng
Published: May 2005

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 × 0.3 × 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which in enzymatically active upon dissolution. These crystals belong to the monoclinic space group P21 and have unit cell parameters a =114.5 Å, b=139.6 Å, c=125.7 Å, β=98.1° Self‐rotation function studies indicate that there are three molecules per asymmetricunit. The crystals diffract to at least 3.0 Å resolution. These are the first crystals of a DNA‐dependent RNA polymerase suitable for high‐resolution X‐ray structure determination.
Three‐dimensional structure of a fluorescein–Fab complex crystallized in 2‐methyl‐2,4‐pentanediol
Authors: Herron James N. , He Xiao‐min , Mason Martha L. , Voss Edward W. , Edmundson Allen B.
Published: May 2005

The crystal structure of a fluorescein–Fab (4‐4‐20) complex was determined at 2.7 Å resolution by molecularreplacement methods. The starting model was the refined 2.7 Å structure of unliganded Fab from an autoantibody (BV04‐01) with specificity for single‐stranded DNA. In the 4‐4‐20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol‐arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2‐3 orders of magnitude in the 4‐4‐20 molecules relative to other members of an idiotypic family of monoclonalantifluorescyl antibodies. The phenyl carboxyl group of fluorescein appearsto be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2‐methyl‐2,4‐pentanediol (MPD), trapped in the interface of the variable domainsjust below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.
Molecular dynamics effects on protein electrostatics
Authors: Wendoloski John J. , Matthew James B.
Published: May 2005

Electrostatic calculations have been carried out on a number of structural conformers of tuna cytochrome c Conformers were generated using molecular dynamics simulations with a range of solvent simulating, macroscopic dielectric formalisms, and one solvent model that explicitly included solvent water molecules. Structures generated using the lowest dielectric models were relatively tight, with‐side chains collapsed on the surface, while those from the higher dielectric modelshad more internal and external fluidity, with surface side chains exploring a fuller range of conformational space. The average structure generated with the explicitly solvated model corresponded most closely with the crystal structure. Individual pK values, overall titration curves, and electrostatic potential surfaces were calculated for average structures and along each simulation. Differences between structural conformers within each simulation give rise to substantial changes in calculated local electrostatic interactions, resulting in pK value fluctuations for individual sites in the protein that very by 0.3–2.0 pK units from the calculated time average. These variations are due to the thermal side chain reorientations that produce fluctuations in charge site separations. Properties like overall titration curves and pH dependent stability are not as sensitive to side chain fluctuations within a simulation, but there are substantial effects between simulation due to markeddifferences in average side chain behavior. These findings underscore the importance of proper dielectric formalism in molecular dynamics simulations when used to generate alternate solution structures from a crystal structure, and suggest that conformers significantly removed from the averagestructure have altered electrostatic properties that may prove important inepisodic protein properties such as catalysis.
A 3D building blocks approach to analyzing and predicting structure of proteins
Authors: Unger Ron , Harel David , Wherland Scot , Sussman Joel L.
Published: May 2005

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of Cα coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set ofstandard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can “replace” about 76% ofall hexamers in well‐refined known proteins with an error of less than 1 Å, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.
Structural analysis of antiviral agents that interact with the capsid of human rhinoviruses
Authors: Badger John , Minor Iwona , Oliveira Macros A. , Smith Thomas J. , Rossmann Michael G.
Published: May 2005

X‐Ray diffraction data have been obtained for nine related antiviral agents (“WIN compounds”) while bound to human rhinovirus 14 (HRV14).These compounds can inhibit both viral attachment to host cells and uncoating. To calculate interpretable electron density maps it was necessary to account for (1) the low (∼60percnt;) occupancies of these compounds in the crystal, (2) the large (up to 7.9 Å) conformational changes induced at the attachment site, and (3) the incomplete diffraction data. Application of a density difference map technique, which exploits the 20‐fold noncrystallographic redundancy in HRV14, resulted in clear images of the HRV14:WIN complexes. A real‐space refinement procedure was used to fit atomic models to these maps.

The binding site of WIN compounds in HRV14 is a hydrophobic pocket composed mainly from residues that form the β‐barrel of VP1. Among rhinoviruses, the residues associated with the binding pocket are far more conserved than external residues and are mostly contained within regular secondary structural elements. Molecular dynamics simulations of three HRV14:WIN complexes suggest that portions of the WIN compounds and viral protein near the entrance of the binding pocket are more flexible than portions deeper within the β‐barrel.
An NMR‐based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution
Authors: de Vlieg J. , Berendsen H. J. C , van Gunsteren W. F.
Published: May 2005

The results of a 125 psec molecular dynamics simulation of a lac headpiece–operator complex in aqueous solution are reported. The complexsatisfies essentially all experimental distance information derived from two‐dimensional nuclear magnetic resonance (2‐D‐NMR) studies. The interaction between lac repressor headpiece and its operator based on many direct‐ and water‐mediated hydrogenbonds and nonpolar contacts which allow the formation of a tight complex. Nostable hydrogen bonds between side chains and bases and found, while specific contacts occur between both nonpolar groups and, to a lesserextent, through water‐mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, andchemically induced dynamic nuclear polarization (CIDNP) data.
Comparing short protein substructures by a method based on backbone torsion angles
Authors: Karpen Mary E. , de Haseth Pieter L. , Neet Kenneth E.
Published: May 2005

An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes Δt, the root mean square difference in ϕ and ψ torsion angles over a small number of amino acids (typically 3–5). Using this algorithm, large number of protein substrates comparisons were feasible. The parameter Δt was sensitive to variations in local protein conformation, and it correlates with Δr, the root mean square deviation in atomic coordinates. Values for Δt were obtained that define similarity thresholds, which determine whether two substructure are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of Δt due to measurement noise fromcomparisons of independently refined coordinates of the same protein. A sample distribution of Δt from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons large numbers of nonmatching comparisons. Unlike methods based on Cα atoms alone, Δt was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologus proteins by Δt showed that the active site torsion angles are highly conserved. The Δt method was applied to the α‐chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.
A computer model to dynamically simulate protein folding: Studies with crambin
Authors: Wilson Charles , Doniach Sebastian
Published: May 2005

The current work describes a simplified representation of protein structure with uses in the simulation of protein folding. The model assumes that a protein can be represented by a freely rotating rigid chain with a single atom approximately the effect of each side chains. Potentials describing the attraction or repulsion between different types of amino acids are determined directly from the distribution of amino acids in the database of known protein structures. The optimization technique of simulated annealinghas been used to dynamically sample the conformations available to this sample model, allowing the protein to evolve from an extended, random coil into a compact globular structure. Many characteristics expected of true proteins, such as the sequence‐dependent formation of secondary structure, the partitioning of hydrophobic residues, and specific disulfide, suggestion the model may accurately simulate the folding process.
Ras interaction with the GTPase‐activating protein (GAP)
Authors: Schaber Michael D. , Garsky Victor M. , Boylan Douglas , Hill Wendy S. , Scolnick Edward M. , Marshall Mark S. , Sigal Irving S. , Gibbs Jackson B.
Published: May 2005

Biologically active forms of Ras complexed to GTP can bind to the GTP ase‐activating protein (GAP), which has been implicated as a possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32–40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser‐35]Ras binds to GAP as effectively as wild‐type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser‐35]Ras can also efficiently stimulate the S. cerevisiae target of Ras adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17–44 and 17–32 competed with the binding of RAS to E. coli‐expressed GAP with IC50 values of 2.4 and 0.9 μM, respectively, whereas Ras peptide 17–26 was without effect up to 400 μM. A related peptide from the yeast GTP‐binding protein YPT1 analogous to Ras peptide 17–32 competed with an IC50 value of 19 μM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17–32 may be important for Ras binding to GAP.
Recombinant HIV1 protease secreted by Saccharomyces cerevisiae correctly processes myristylated gag polyprotein
Authors: Pichuantes Sergio , Babé Lilia M. , Barr Philip J. , Craik Charles S.
Published: May 2005

The protease of the human immunodeficiency virus type I (HIV1) was expressed both intracellularly and extracellularly in Saccharomyces cerevisiae. Intracellular expression of the protease was achieved by fusing a 179 amino acid precursor form of the protease to human superoxide dismutase (hSOD). Self‐processing of the viral enzyme from the hybrid precursor was demonstrated to ocur within the yeast host. Secretion of the protease was achieved by fusing the leader sequence of yeast α‐factor to the precursor form of the protrease or to the 99 amino acid mature form of the protease. Authentic and active forms of the retroviral enzyme were detected in yeast supernatants of cells expressing the precursor or the mature form of the protease. A D25E active site variant of the retroviral enzyme exhibited diminished autocatalytic activity when expressed intracellularly or secreted from yeast. The wild‐type protease was active in an in vitro assay on the natural substrate, myristylated gag precursor, Pr53gag. Correct processing of Pr53gag at the Tyr 138–Pro 139 junction was confirmed by amino terminal sequence analysis of the resulting capsid protein (CA, p24). The secreted protease was purified to homogeneity from yeast media using preparative isoelectric focusing and reverse‐phase HPLC. Amino terminal sequence analysis showed a sequence beginning at amino acid 1 of the mature enzyme (Pro) and another sequence beginning at amino acid 6 (Trp). This shorter sequence may represent a natural autolytic product of the proteaseaspartyl protease.
Axial ligand replacement in horse heart cytochrome c by semisynthesis
Authors: Raphael Adrienne L. , Gray Harry B.
Published: May 2005

Semisynthesis has been employed to replace the axial methionine in horse heart cytochrome c with histidine. The reduction potential of the His‐80 protein (cyt c‐His‐80) is 41 mV vs NHE (0.1 M phosphate; pH 7.0; 25°C). The absorption spectra of oxidized and reduced cyt c‐His‐80 are very similar to those of the native protein in the porphyrin region, but the 695 nm band is absent in the oxidized His‐80 protein.
Alternate succession of steps can lead to the folding of a multidomain oligomeric protein
Authors: Murry‐Brelier Anne , Goldberg Michel E.
Published: May 2005

The β2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured form of β2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of β chains. We describe the kinetics of regain of a variety of physical, functional, ad immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded β2. It is shown that whereas identical molecular events over with the same kinetics, the two folding pathways are different, and involve different structural intermediates.
The surface area of monomeric proteins: Significance of power law behavior
Authors: Bryant Stephen H. , Islam Suhail A. , Weaver David L.
Published: May 2005

The coefficients in a power low fit of accessible area versus molecular weight for high‐reslution monomeric protein structures are assessed with respect to statistical accuracy using bootstrap analyses, and with respect to physical significance using model systems and the concept of roughness or fractal structure of the protein surface.
On the nature of antibody combining sites: Unusual structural features that may confer on these sites an enhanced capacity for binding ligands
Authors: Padlan Eduardo A.
Published: May 2005

A detailed analysis of the structural aspects of antibody–antigen interactions has been made possible by the availability of X‐ray structures for three complexes of antilysozyme Fabs to lysozyme (reviewed by Davies et al: J. Biol. Chem. 263:10541–10544, 1988.) Examination of the antigen‐contacting residues in the three antilysozyme Fabs reveals the occurrence of a large number of aromatics, particularly tyrosines, and the absence of apolar, aliphatic residues. Calculation of the frequency of occurrence of the various amino acids types reveals that tyrosines are three times, and histidines and asparagines eight times, more likely to be found in the complementarity‐determinig regions that in the framework of the variability of the residue in FVs (the modules containing variable domains of the light and heavy chains) of known three‐dimensional structure indicates that tryosines and tryptophans are more exposed when they occur in the complementarity‐determining regions that when in the framework. Furthermore, many more of the asparagines in the complementarity‐determining regions than in the framework are buried. These aspararagines appear to have a structural role in that they hydrogen bond through their side chains to other side chains and, Even more so, to the protein backbone. The stabilizing effect of the asparragines, plus the rigidity of the framework, may serve to allow the greater exposure of the aromatic residues to solvent. In view of the greater potential contribution of aromatic side chains to the total binding energy, these results suggest that antibody‐combining sites have structural features that make them especially studied for interacting with lagands.
β‐Lactamase of Bacillus licheniformis 749/C at 2 Å resolution
Authors: Moews Paul C. , Knox James R. , Dideberg Otto , Charlier Paulette , Frère Jean‐Marie
Published: May 2005

Two crystal (A and B) of the 29,500 Da Class A β‐lactamase (penicillinase) from Bacillus licheniformis 749/C have been examined crystallographically. The structure of B‐form crystals has been solved to 2 Å resolution, the starting model for which was a 3.5 Å structure obtained from A‐form crystals. The β‐lactamase has an α + β structure with 11 helices and 5 β‐strands seen also in a peinicilin target DD‐peptidase of Streptomyces R61.1 Atomic parameters of the two molecules in the asymmetric unit were refined by simulated annealing at 2.0 Å resolution. The R factor is 0.208 for the 27,330 data greater than 3 (F), with water molecules excluded from the model. The catalytic Ser‐70 is at the N‐terminus of a helix and is within hydrogen bonding distance of conversed Lys‐73. Also interacting with the Lys‐73 are Asn‐132 and the conserved Glu‐166, which is on a potentially flexible helix‐containing loop. The structure suggests the binding of β‐lactam substrates is facilitated by interactions with Lys‐234, Tyhr‐235, and Ala‐237 in a conserved β‐strand peptide, which is antiparallel to the β‐lactam's acylamido linkage; an exposed cavity near Asn‐170 exits for acylamido Substituent. The reactive double bond of clavulanate‐type inhibitors may interact with Arg‐244 on the fourth β‐strand. A very similar binding site architecture is seen in the DD‐peptidase.
Molecular dynamics study of secondary structure motion in proteins: Application to myohemerythrin
Authors: Rojewska Danuta , Elber Ron
Published: May 2005

The concept of secondary structure motions is examined in a molecular dynamics simulation of the protein myohemerythrin. We extracted from the simulation a corresponding trajectory of helices and demonstrated that the fluctuations of the protein are dominated by a rigid shift of these secondary structure elements. The relative motions of the helices are regular, with no clear periodicity. They are bounded by ∼2 for the center of mass motions and by 20° for the relative orientations. The potential of mean force for the interactions of the helices was calculated, and the correlations between the different extended motions were investigated. It shown that the one‐dimensional mean force potentials are close to quadratic for most of the helices coordinates. The anharmonicity is reflected by changes in the direction of the normal modes as a function of the energy and by the existence of multiple free energy minima for the helices packing. The multiple conformations are associated with a single type of secondary structure coordinate: the angle that describes the relative orientation of the helices in a plane perpendicular to the line connecting their center of mass.
Bundles of amphipathic transmembrane α‐helices as a structural motif for ion‐conducting channel proteins: Studies on sodium channels and acetylcholine receptors
Authors: Oiki Shigetoshi , Madison Vincent , Montal Mauricio
Published: May 2005

Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage‐sensitive sodium channel and the nicotinic cholinergic receptor. We report low‐energy arrangements of α‐helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion‐conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3–5 amphipathic α‐helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar‐neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area ∼ 16 Å2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of α‐helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area ∼ 46 Å2). Thus, bundles of amphipathic α‐helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.
Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase
Authors: Rose John P. , Hempel John , Kuo Ingrid , Lindahl Ronald , Wang Bi‐Cheng
Published: May 2005

NAD‐linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 A1DH (from an Escherichia coli expression system) suitable for X‐ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 × 0.3 × 0.2 mm, diffract to 2.5 Å resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 Å, b = 170.67 Å, c = 47.15 Å, and β = 110.5°. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self‐rotation function study produced significant rotation peaks (58% of the origin) on the K = 180 section at ψ = 90° and ϕ = 71° and 341°, indicating that the pseudo‐dimer axis is (or is very nearly) perpendicular to the b‐axis.
A helix–turn–strand structural motif common in α–β proteins
Authors: Rice Phoebe A. , Goldman Adrian , Steitz Thomas A.
Published: May 2005

By exhaustive structural comparisons, we have found that about one‐third of the α‐helix–turn–β–strand polypeptides in α–β barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the α‐helix and the β‐strand is somewhat constrained, and second, the β‐strand contains a hydrophobic patch that fits into a hydrophobic pocket on the α‐helix. The geometry of the turn does not seem to be a major determinant of the α–β unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn–for instance, when the turns between the α‐helix and the β‐strands are very long. It also occurs much less frequently in flat‐sheet α–β proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which α–β barrels are constructed.