Showing papers in "Proteins in 1989"

The neuraminidase of influenza virus.

Abstract: It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.

A 3D building blocks approach to analyzing and predicting structure of proteins

Abstract: 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 A, 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

The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+, and the inhibitor pdTp, refined at 1.65 A.

Abstract: The structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3',5'-bisphosphate (pdTp) has been refined by stereochemically restrained least-squares minimization to a crystallographic R value of 0.161 at 1.65 A resolution. The estimated root-mean-square (rms) error in the coordinates is 0.16 A. The final model comprises 1082 protein atoms, one calcium ion, the pdTp molecule, and 82 solvent water molecules; it displays an rms deviation from ideality of 0.017 A for bond distances and 1.8 degrees for bond angles. The mean distance between corresponding alpha carbons in the refined and unrefined structures is 0.6 A; we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket. The details of the calcium liganding and solvent structure in the active site are clearly shown in the final electron density map. The structure of the catalytic site is consistent with the mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry-related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that of enzyme and substrate.

The effects of truncating long-range forces on protein dynamics

Abstract: This paper considers the effects of truncating long-range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long-range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom-atom truncation based on distance; (4) simple atom-atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group-group truncation based on distance; and (6) simple group-group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates, the rms deviation from the average coordinates of a no cutoff simulation and from the X-ray structure of the protein. The simulations in which long-range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 A, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5-9 A range gives reasonable results for a distance-dependent dielectric, but not using a constant dielectric. Both the atom-atom and group-group truncation methods based on distance shows large rms deviation and fluctuation for short cutoff distances, while for cutoff distances of 11 A or greater, reasonable results are achieved. Although comparison of these to distance-based truncation methods show surprisingly larger rms deviations for the group-group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom-atom or group-group list-based simulations generally appear to be less stable than the distance-based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.

Three‐dimensional structure of a fluorescein–Fab complex crystallized in 2‐methyl‐2,4‐pentanediol

Abstract: The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A 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 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to 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 domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

Evolution of CuZn superoxide dismutase and the Greek Key β‐barrel structural motif

Abstract: Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key beta-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. The beta-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the beta-strands and at both termini. Thus, the beta-barrel with only four invariant residues is apparently over-determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility of the Greek key beta-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.

The structure of aconitase

Abstract: The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.

A computer model to dynamically simulate protein folding: Studies with crambin

Abstract: 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 approximating the effect of each side chain. 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 annealing has been used to dynamically sample the conformations available to this simple 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 pairing, are reproduced by the simulation, suggesting the model may accurately simulate the folding process.

Structural principles of α/β barrel proteins: The packing of the interior of the sheet

Abstract: Alpha/beta barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight-stranded beta-sheets and the packing of the residues at the center of the barrel. The packing in this region is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at some of the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues from the second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even-numbered strands that form the central layer, and side chains from the odd-numbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed beta barrel.

A protein structural motif that bends DNA.

Abstract: The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli, and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high-resolution structure of HU, we propose a model for this interaction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.

Further studies of the helix dipole model: Effects of a free α‐NH3+ or α‐COO− group on helix stability

Abstract: Interactions between the α-helix peptide dipoles and charged groups close to the ends of the helix were found to be an important determinant of α-helix stability in a previous study.1 The charge on the N-terminal residue of the C-peptide from ribonuclease A was varied chiefly by changing the α-NH2 blocking group, and the correlation of helix stability with N-terminal charge was demonstrated. An alternative explanation for some of those results is that the succinyl and acetyl blocking groups stabilize the helix by hydrogen bonding to an unsatisfied main-chain NH group. The helix dipole model is tested here with peptides that contain either a free α-NH α-COO− groups, and no other charged groups that would titrate with similar pKa's. This model predicts that α-NH3α-COO- groups are helix-destabilizingand that the destabilizing interactions are electrostatic in origin. The hydrogen bonding model predicts that α-NH3 and α-COO- groups are not themselves helix-destabilizing, but that an acetyl or amide blocking group at the N- or C- terminus, respectively, stabilizes the helix by hydrogen bonding to an unsatisfied main-chain NH or CO group. The results are as follows: (1) Removal of the charge from α-NH3 and α-COO- groups by pH titration stabilizes an α-helix. (2) The increase in helix stability on pH titration of these groups is close to the increase produced by adding an acetyl or amide blocking group. (3) The helix-stabilizing effect of removing the charge from α-NH3 and α-COO- groups by pH titration is screened by increasing the NaCl concentration, and therefore the effect is electrostatic in origin. (4) Replacing the C-terminal amide blocking group with a methylester blocking group, which cannot donate a hydrogen bond, causes little change in helix stability.

Describing Protein Structure: A General Algorithm Yielding Complete Helicoidal Parameters and a Unique Overall Axis

Abstract: We present a general and mathematically rigorous algorithm which al- lows the helicoidal structure of a protein to be calculated starting from the atomic coordinates of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal pa- rameters describing the location of each pep- tide unit. The parameters obtained form a com- plete and independent set and can therefore be used for analyzing, comparing, or reconstruct- ing protein backbone geometry. This algorithm has been implemented in a computer program named P-Curve. Several examples of its possi- ble applications are discussed.

Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation.

Abstract: Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix. After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.

Engineering subtilisin BPN' for site-specific proteolysis.

Abstract: A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

Effect of protein conformation on rate of deamidation: Ribonuclease A

Abstract: The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.

Amino acid substitutions that increase the thermal stability of the λ Cro protein

Abstract: A mutant Cro protein, which bears the Ile-30 Leu substitution, is thermally unstable and degraded more rapidly than wildtype Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12°C to 14°C. These amino acid substitutions affect residues 16 and 26, which are substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild-type Cro.

Structural determinants of the conformations of medium-sized loops in proteins.

Abstract: Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites. The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen-bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen-binding loops in immunoglobulins, we identified medium-sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations. For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward-pointing main chain atoms. For oops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein. The following picture emerges: Medium-sized lops of similar conformation are stabilized by similar interaction. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles.

Abstract: Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ras interaction with the GTPase-activating protein (GAP).

Abstract: Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as 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 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM 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.

Structural basis of hierarchical multiple substates of a protein. I: Introduction.

Abstract: 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.

Rebuilding flavodoxin from Cα coordinates: A test study

Abstract: The tertiary structure of flavodoxin has been model built from only the X-ray crystallographic alpha-carbon coordinates. Main-chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 A across all atoms to the native structure. Regular secondary structural elements were modeled more accurately than other regions. About 40% of the chi 1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions. The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contacts, and hydrogen-bonding patterns could identify poorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.

Axial ligand replacement in horse heart cytochrome c by semisynthesis.

Abstract: 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 degrees 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.

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy.

Abstract: With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.

Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli

Abstract: A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala-13 to Gly-185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short-lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species isonly partially processed, and it, a 13 kDA intermediate, and the mature 11 kDA enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2- and COOH-terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2≅ 9 min) processes itself into a species with a mass of ∼13kDa and a species with a mass of ∼11 kDa. Both of these latter species can be separated by RP-HPLC, have the NH2-terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP-HPLC and SDS poly acrylamide gel electrophoresis. On extended incubation at 4°C at either neutral or acidic pH all species of the proteinexhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.

The construction of new proteins: V. A template‐assembled synthetic protein (TASP) containing both a 4‐helix bundle and β‐barrel‐like structure

Abstract: The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a beta-barrel as two folding "domains" is described. For the de novo design of proteins, amphiphilic helices (alpha) and beta-sheets (beta) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intramolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4 alpha)(4 beta). 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.

Use of restrained molecular dynamics in water to determine three‐dimensional protein structure: Prediction of the three‐dimensional structure of Ecballium elaterium trypsin inhibitor II

Abstract: Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution.

Abstract: The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies 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 is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, 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, and chemically induced dynamic nuclear polarization (CIDNP) data.

Rapid calculation of the solution scattering profile from a macromolecule of known structure.

Abstract: If one expands the structure factor equation in spherical coordinates, rotational averaging of the molecular Fourier transform, which leads directly to the solution scattering profile, is greatly simplified. It becomes a projection in the polar and azimuthal angular variables. The profile is given by The index j runs over all atoms; r, θ, ϕ are atomic coordinates and e and N are constants; the Ym,n are complex spherical harmonics, and Jn are spherical Bessel functions; R = 2 sin θ/λ. The effects of solvent have been modeled by subtracting from each protein atom a properly weighted water. Hydrogens have been included by using scattering curves fj derived from the spherical averaging ofprotein atoms with their attached hydrogens. This approach may also be satisfactory for neutron scattering. Published scattering profiles2 for lysozyme and BPTI have been accurately matched in less than one-tenth the time required by other methods. Separate, adjustable temperature factors for the protein, solvent waters, and bound watersare used, and appear to be needed. In the case of BPTI, as suggested by NMR observations, the observed diffraction pattern was much better accounted for by including only 4 tightly bound waters rather than the roughly 60 seen by crystallography.