Stereochemically constrained peptides. Theoretical and experimental studies on the conformations of peptides containing 1-aminocyclohexane carboxylic acid

Conformational energy calculations on the model system N-acetyl- 1 -aminocyclohexanecarboxylic acid N'methylamide (Ac-Acc6-NHMe), using an average geometry derived from 13 crystallographic observations, establish that the Acc6 residue is constrained to adopt conformations in the helical regions of In contrast, the a,a-dialkylated residue with linear hydrocarbon side chains, a,a-di-n-propylglycine favors fully extended backbone structures (6 1= $ = 180'). The crystal structures of two model peptides, Boc-(Acc6),-OMe (type 111 @-turn at -Acc6(1)-Acc6(2)-) and Boc-Pro-Acc6-Ala-OMe (type I1 P-turn at -Pro-Acc6-), establish that Acc6 residues can occupy either position of type 111 P-turns and the i + 2 position of type I1 @-turns. The stereochemical rigidity of these peptides is demonstrated in solution by NMR studies, which establish the presence of one intramolecular hydrogen bond in each peptide in CDCI, and (CDJ2S0. Nuclear Overhauser effects permit characterization of the @-turn conformations in solution and establish their similarity to the solid-state structures. The implications for the use of Acc6 residues in conformational design are considered.

Aqueous channels within apolar peptide aggregates. Solvated helix of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib- -Ala-Leu-Aib-OMe. H2O.CH3OH

Although the peptide Boc-Aibl-Ala2-Leu3- Aib4-Alas Leu'-Aib7-Ala8-Leu9-Aib'0-OMe [with a t-butoxycarbonyl(Boc) blocking group at the amino terminus, a methyl ester (OMe) at the carboxyl terminus, and four a-aminoisobutyric (Aib) residues] has a 3-fold repeat of residues, the helix formed by the peptide backbone is irregular. The carboxyl-terminal half assumes an at-helical form with torsion angles ) and r of approximately -60° and -45°, respectively, whereas the amino-terminal half is distorted by an insertion of a water molecule between the amide nitrogen of Ala5 [N(5)] and the carbonyl oxygen of Ala2 [0(2)]. The water molecule W(1) acts as a bridge by forming hydrogen bonds N(5).W(1) (2.93 A) and W(1)---0(2) (2.86 A). The distortion of the helix exposes the carbonyl oxygens of Aib' and Aib4 to the outside environment, with the consequence that the helix assumes an amphiphilic character despite having all apolar residues. Neighboring helices in the crystal run in antiparallel directions. On one side of a helix there are only hydrophobic contacts with efficient interdigitation of leucine side chains with those from the neighboring helix. On the other side of the helix there are hydrogen bonds between protruding carbonyl oxygens and four water molecules that separate two neighboring helices. Along the helix axis the helices bind head-to-tail with a direct hydrogen bond N(2)-0(9) (3.00 A). Crystals grown from methanol/water solution are in space group P2, with a = 15.778 ± 0.004 A, b = 11.228 ± 0.002 A, c = 18.415 ± 0.003 A, = 102.10 ± 0.02ur and two formula units per cell for C49HON1003 2H2OCH3OH. The overall agreement factorR is 7.5% for 3394 reflections observed with intensities >3a(F), and the resolution is 0.90 A.

Rigid and flexible region in lysozyme and the invariant features in its hydration shell

Water-mediated transformations provide a useful handle for exploring the flexibility in protein molecules and the invariant features in their hydration shells. Low-humidity monoclinic hen egg white lysozyme, resulting from such a transformation, has perhaps the lowest solvent content observed in any protein crystal so far and has a well-ordered structure. A detailed comparison involving this structure, low-humidity tetragonal lysozyme, and the other available refined crystal structures of the enzyme permits the delineation of the relatively rigid, moderately flexible and highly flexible regions of the molecule. The relatively rigid region forms a contiguous structural unit close to the molecular centroid and encompasses parts of of the main beta-structure and three alpha-helices. The hydration shell of the protein contains 30 invariant water molecules. Many of them are involved in holding different parts of the molecule together or in stabilizing local structure. Five of the six invariant water molecules attached to the substrate-binding region form part of a water cluster contiguous with the side-chains of the catalytic residues Glu-35 and Asp-52.

Protein Hydration and Water Structure: X-ray Analysis of a Closely Packed Protein Crystal with Very Low Solvent Content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding.

Analysis of the effects of amino-acid-sequence on the structure of proteins

The conformation of amino acid side chains as observed in well-determined structures of globular proteins has earlier been extensively investigated. In contrast, the structural features of the polypeptide backbone that result from the occurrence of specific amino acids along the polypeptide have not been analysed. In this article, we present the statistically significant features in the backbone geometry that appear to be a consequence of the occurrence of rotamers of different amino acid side chains by analysing 102 well-refined structures that form a random collection of proteins. It is found that the persistence of helical segments around each residue is influenced by the residue type. Several residues exert asymmetrical influence between the carboxyl and amino terminal polypeptide segments. The degree to which secondary structures depart from an average geometry also appears to depend on residue type. These departures are correlated to the corresponding Chou and Fasman parameters of amino acid residues. The frequency distribution of the side chain rotamers is influenced by polypeptide secondary structure. In turn, the rotamer conformation of side chain affects the extension of the secondary structure of the backbone. The strongest correlation is found between the occurrence of g+ conformation and helix propagation on the carboxyl side of many residues.

Characterization of substrate UpA binding to RNase A--computer modelling and energetics approach.

In the past two decades RNase A has been the focus of diverse investigations in order to understand the nature of substrate binding and to know the mechanism of enzyme action. Although this system is reasonably well characterized from the view point of some of the binding sites, the details of interactions in the second base binding (B2) site is insufficient. Further, the nature of ligand-protein interaction is elucidated generally by studies on RNase A-substrate analog complexes (mainly with the help of X-ray crystallography). Hence, the details of interactions at atomic level arising due to substrates are inferred indirectly. In the present paper, the dinucleotide substrate UpA is fitted into the active site of RNase A Several possible substrate conformations are investigated and the binding modes have been selected based on Contact Criteria. Thus identified RNase A-UpA complexes are energy minimized in coordinate space and are analysed in terms of conformations, energetics and interactions. The best possible ligand conformations for binding to RNase A are identified by experimentally known interactions and by the energetics. Upon binding of UpA to RNase A the changes associated,with protein back bone, Side chains in general and at the binding sites in particular are described. Further, the detailed interactions between UpA and RNase A are characterized in terms of hydrogen bonds and energetics. An extensive study has helped in interpreting the diverse results obtained from a number of experiments and also in evaluating the extent of changes the protein and the substrate undergo in order to maximize their interactions.

Metallo-beta-lactamase-Catalyzed Hydrolysis of Cephalosporins: Some Mechanistic Insights into the Effect of Heterocyclic Thiones on Enzyme Activity

The hydrolysis of beta-lactam antibiotics using zinc-containing metallo-beta-lactamases (m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic - SR side chains are less prone to m beta l-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MU, MDT, DMETT, and MMA, the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic - SR moieties eliminated from the beta-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms, these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear zinc(II) thiolate complexes indicates that the -SR side chains eliminated from the antibiotics may interact with the zinc(II) metal center of m beta l through their sulfur atoms.

One protein�many functions

The concept of one enzyme-one activity had influenced biochemistry for over half a century. Over 1000 enzymes are now described. Many of them are highly 'specific'. Some of them are crystallized and their three-dimensional structures determined. They range from 12 to 1000 kDa in molecular weight and possess 124 to several hundreds of amino acids. They occur as single polypeptides or multiple-subunit proteins. The active sites are assembled on these by appropriate tertiary folding of the polypeptide chain, or by interaction of the constituent subunits. The substrate is held by the side-chains of a few amino acids at the active site on the surface, occupying a tiny fraction of the total area. What is the bulk of the protein behind the active site doing? Do all proteins have only one function each? Why not a protein have more than one active site on its large surface? Will we discover more than one activity for some proteins? These newer possibilities are emerging and are finding experimental support. Some proteins purified to homogeneity using assay methods for different activities are now recognized to have the same molecular weight and a high degree of homology of amino acid sequence. Obviously they are identical. They represent the phenomenon of one protein-many functions.

Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex. We have substituted the wild-type residue at position 13, methionine (Met), with norleucine (Nle), where the only covalent change is the replacement of the sulfur atom with a methylene group. The thermodynamic parameters associated with the binding of this variant to S-protein, determined by titration calorimetry in the temperature range 10-40 degrees C, are reported and compared to values previously reported [Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426] for other position 13 analogs. The differences in the free energy and enthalpy of binding between the Met and Nle peptides are 0.6 and 7.9 kcal/mol at 25 degrees C, respectively. These differences are slightly larger than, but comparable to, the differences in the values for the Met/Ile and Met/Leu pairs. The structure of the mutant complex was determined to 1.85 Angstrom resolution and refined to an R-factor of 17.4% The structures of mutant and wild-type complexes are practically identical although the Nle side chain has a significantly higher average B-factor than the corresponding Met side chain. In contrast, the B-factors of the atoms of the cage of residues surrounding position 13 are all somewhat lower in the Nle variant than in the Met wild-type. Thus, the large differences in the binding enthalpy appear to reside entirely in the difference in chemical properties or dynamic behavior of the -S- and -CH2- groups and not in differences in the geometry of the side chains or the internal cavity surface. In addition, a novel method of obtaining protein stability data by means of isothermal titration calorimetry is introduced.

The stability of transmembrane helices: A molecular dynamics study on the isolated helices of bacteriorhodopsin

Bacteriorhodopsin (bR) continues to be a proven testing ground for the study of integral membrane proteins (IMPs). It is important to study the stability of the individual helices of bR, as they are postulated to exist as independently stable transmembmne helices (TMHs) and also for their utility as templates for modeling other IMPs with the postulated seven-helix bundle topology. Toward this purpose, the seven helices of bR have been studied by molecular dynamics simulation in this study. The suitability of using the backbone-dependent rotamer library of side-chain conformations arrived at from the data base of globular protein structures in the case TMHs has been tested by another set of ? helix simulations with the side-chain orientations taken from this library. The influence of the residue's net charge oil the helix stability was examined by simulating the helices III, IV, and VI (from both of the above sets of helices) with zero net charge on the side chains. The results of these 20 simulations demonstrate in general the stability of the isolated helices of bR in conformity with the two-stage hypothesis of IMP folding. However, the helices I, II, V, and VII are more stable than the other three helices. The helical nature of certain regions of III, IV, and VI are influenced by factors such as the net charge and orientation of several residues. It is seen that the residues Arg, Lys, Asp, and Glu (charged residues), and Ser, Thr, Gly, and Pro, play a crucial role in the stability of the helices of bR. The backbone-dependent rotamer library for the side chains is found to be suitable for the study of TMHs in IMP. (C) 1996 John Wiley & Sons, Inc.

Role of Water in the Enzymatic Catalysis: Study of ATP plus AMP -> 2ADP Conversion by Adenylate Kinase

The catalytic conversion ATP + AMP -> 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP. molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a. phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate stale of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for. the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

The structures of bacteriorhodopsin with different retinal-Schiff base orientations--computer modeling and energy minimization studies

Bacteriorhodopsin has been the subject of intense study in order to understand its photochemical function. The recent atomic model proposed by Henderson and coworkers based on electron cryo-microscopic studies has helped in understanding many of the structural and functional aspects of bacteriorhodopsin. However, the accuracy of the positions of the side chains is not very high since the model is based on low-resolution data. In this study, we have minimized the energy of this structure of bacteriorhodopsin and analyzed various types of interactions such as - intrahelical and interhelical hydrogen bonds and retinal environment. In order to understand the photochemical action, it is necessary to obtain information on the structures adopted at the intermediate states. In this direction, we have generated some intermediate structures taking into account certain experimental data, by computer modeling studies. Various isomers of retinal with 13-cis and/or 15-cis conformations and all possible staggered orientations of Lys-216 side chain were generated. The resultant structures were examined for the distance between Lys-216-schiff base nitrogen and the carboxylate oxygen atoms of Asp-96 - a residue which is known to reprotonate the schiff base at later stages of photocycle. Some of the structures were selected on the basis of suitable retinal orientation and the stability of these structures were tested by energy minimization studies. Further, the minimized structures are analyzed for the hydrogen bond interactions and retinal environment and the results are compared with those of the minimized rest state structure. The importance of functional groups in stabilizing the structure of bacteriorhodopsin and in participating dynamically during the photocycle have been discussed.

Synthesis and biological evaluation of novel 2-aralkyl-5-substituted-6-(4 `-fluorophenyl)-imidazo2,1-b]1,3,4]thiadiazole derivatives as potent anticancer agents

Levamisole, the imidazo2,1-b]thiazole derivative has been reported as a potential antitumor agent. In the present study, we synthesized, characterized and evaluated biological activity of its novel analogues with substitution in the aralkyl group and on imidazothiadiazole molecules with same chemical backbone but different side chains namely 2-aralkyl-6-(4'-fluorophenyl)-imidazo2,1-b]1,3,4]thiadiazoles (SCR1), 2-aralkyl-5-bromo-6-(4'-fluorophenyl)-imidazo2,1-b]1,3,4]-thiadiaz oles (SCR2), 2-aralkyl-5-formyl-6-(4'-fluorophenyl)-imidazo2,1-b]1,3,4]-thiadia zoles (SCR3) and 2-aralkyl-5-thiocyanato-6-(4'-fluorophenyl)-imidazo2,1-b]1,3,4]-th iadiazoles (SCR4) on leukemia cells. The cytotoxic studies showed that 3a, 4a, and 4c exhibited strong cytotoxicity while others had moderate cytotoxicity. Among these we chose 4a (IC50, 8 mu M) for understanding its mechanism of cytotoxicity. FACS analysis in conjunction with mitochondrial membrane potential and DNA fragmentation studies indicated that 4a induced apoptosis without cell cycle arrest suggesting that it could be used as a potential chemotherapeutic agent. (C) 2011 Elsevier Masson SAS. All rights reserved.

Intercalative pyrimido[4',5':4,5]thieno(2,3-b)quinolines induce apoptosis in leukemic cells: a comparative study of methoxy and morpholino substitution

DNA intercalating molecules are promising anticancer agents. Polycyclic aromatic molecules such as ellipticine intercalate into double-stranded DNA and affect major physiological functions. In the present study, we have characterized two molecules with the same chemical backbone but different side chains, namely 8-methoxy pyrimido[4',5':4,5]thieno (2,3-b)quinoline-4(3H)-one (MPTQ) and 4-morpholino pyrimido[4',5':4,5]thieno(2,3-b)quinoline (morpho-PTQ) at the 8th and 4th position, respectively. Although both MPTQ and morpho-PTQ show similar biophysical properties with high DNA affinity, here we show that they differ in their biological activities. We find that MPTQ is many fold more potent than morpho-PTQ and is cytotoxic against different leukemic cell lines. IC(50) value of methoxy PTQ was estimated between 2-15 A mu M among the leukemic cells studied, while it was more than 200 A mu M when morpho-PTQ was used. Cell cycle analysis shows an increase in sub-G1 phase, without any particular cell cycle arrest. Annexin V staining in conjunction with comet assay and DNA fragmentation suggest that MPTQ induces cytotoxicity by activating apoptosis. Thus the observed low IC(50) value of MPTQ makes it a promising cancer chemotherapeutic agent.

Phase Characterization and Study of Molecular Order of a Three-Ring Mesogen by (13)C NMR in Smectic C and Nematic Phases

Molecules exhibiting a thermotropic liquid-crystalline property have acquired significant importance due to their sensitivity to external stimuli such as temperature, mechanical forces, and electric and magnetic fields. As a result, several novel mesogens have been synthesized by the introduction of various functional groups in the vicinity of the aromatic core as well as in the side chains and their properties have been studied. In the present study, we report three-ring mesogens with hydroxyl groups at one terminal. These mesogens were synthesized by a multistep route, and structural characterization was accomplished by spectral techniques. The mesophase properties were studied by hot-stage optical polarizing microscopy, differential scanning calorimetry, and small-angle X-ray scattering. An enantiotropic nematic phase was noticed for lower homologues, while an additional smectic C phase was found for higher homologues. Solid-state high-resolution natural abundance (13)C NMR studies of a typical mesogen in the solid phase and in the mesophases have been carried out. The (13)C NMR spectrum of the mesogen in the smectic C and nematic phases indicated spontaneous alignment of the molecule in the magnetic field. By utilizing the two-dimensional separated local field (SLF) NMR experiment known as SAMPI4, (13)C-(1)H dipolar couplings have been obtained, which were utilized to determine the orientational order parameters of the mesogen.