Molecular Dynamics Studies On Nucleoside 2′,3′-Cyclic Phosphates

Abstract

2′,3′-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3′-GMP, 3′-UMP, A>p, G>p, U>p, C>p, GpA>p and UpA>p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.     

Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation

The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.     

5-Nitrouridine-monohydrate: crystal structure and conformation.

The crystal structure of 5-nitrouridine was determined by X-ray analysis. The pyrimidine ring is slightly non-planar, showing a shallow boat conformation. The nitro group has no influence on the C4 - O4 bond length as compared to uridine. The ribose shows the C3'-endo conformation and the base is in the anti orientation to the sugar with a torsion angle of 25.6 degrees. This conformation is stabilized by a hydrogen bond from the base to the ribosyl moiety (H6 ... 05'). Stacking interactions between neighboring bases are almost negligible in the crystal. A water molecule is involved in a bifurcated donating hydrogen bond to 04 and to 052 of the nitro group of the one base and an accepting bond from the H3 of the other base. Two more hydrogen bonds are formed between the water molecule and the ribose. The structural aspects of 5-nitrouridine are discussed with respect to the special stacking features found for 5-nitro-1-(beta-D-ribosyluronic acid)-uracil monohydrate in the crystal (1).     

Models for the 3(10)-helix/coil,pi-helix/coil,and alpha-helix/3(10)-helix/coil transitions in isolated peptides.

Models for the 3(10)-helix/coil and pi-helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation. The models for alpha-helix formation and 3(10)-helix formation have also been combined to describe a three-state equilibrium in which alpha-helical, 3(10)-helical, and coil conformations are populated. The results are compared with the modified Lifson-Roig theory for the alpha-helix/coil equilibrium. The comparison accounts for the experimental observations that 3(10)-helices tend to be short and pi-helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices.     

Conformation of the polar headgroup of sphingomyelin and its analogues

The conformation of the polar headgroup of synthetic D-erythro-stearoylsphingomyelin (1), its L-threo-isomer (2) and phosphorothioyl analogues of 1 (3 and 4) has been studied in detail by high-resolution NMR spectroscopy. In both monomeric and aggregated states the phosphocholine function of 1 adopts the synclinal conformation (alpha 5 torsional angle), in analogy with phosphatidylcholine (Hauser, H., Guyer, W., Pascher, I., Skrabal, P. and Sundell, S. (1980) Biochemistry 19, 366-373). The conformation about the C1-C2 bond (theta 1 angle) of the sphingosine backbone is predominantly -synclinal, analogously to the conformation of the crystalline galactosyl cerebroside (Pascher, I. and Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191). In contrast, the L-threo-isomer displays unrestricted rotation about C1-C2 bond. The possibility of the existence of a hydrogen bond between the 3-hydroxyl function and the bridged oxygen atom of sphingosine responsible for the different conformation of 1 and 2 is discussed. The modification of the phosphate function in 1 with sulfur has no significant effect on the conformation of the resulting analogues. The conformation of all studied compounds about the C-O phosphoester bonds (alpha 1 and alpha 4 torsion angles) is mainly antiperiplanar. Similar to other double-chain phospholipids, sphingomyelin shows a preference towards the antiperiplanar conformation about the C2-C3 bond.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Pyridine interactions with phenolic groups in water: evidence for hydrogen bonding and hydrophobic association

Pyridine interactions with phenol, substituted phenol, tyrosine and poly(Glu50,Tyr50) in aqueous solutions have been studied by ultraviolet (UV) difference spectroscopy, spectrophotometric pH titration, circular dichroism (CD) and proton magnetic resonance (PMR) spectroscopy. A red shift and spectral sharpening of the near-UV spectrum of phenol in water was noted at pyridine concentrations greater than 0.25 M. In addition, the spectrophotometric equivalence point for the phenol- or substituted phenol-phenolate equilibrium was increased about 0.5 pH units upon the addition of 1.0 M pyridine. PMR studies were consistent with the formation of a 1 : 1 phenol-pyridine hydrogen bonded complex. The equilibrium constant derived for this interaction, 0.6-0.7 M-1, is greater than the corresponding value for phenol-acetate hydrogen bonding in water. Enhancement of thepyridine hydrogen bond interaction with Tyr within poly(Glu50,Tyr50) was observed at pH greater than 12 due to a hydrophobic microenvironment produced by pyridine molecules intercalating between neighboring tyrosyl residues.     

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.     

3-Ribosyl-6-methyluracil as a phosphate acceptor in the synthesis of the internucleotide bond catalyzed by pancreatic ribonuclease.

The synthesis of uridylyl-(3'-5')-3-ribosyl-6-methyluracil (UprmU) catalyzed by pancreatic ribonuclease (EC 3.4.1.22) has been performed using uridine 2', 3'-cyclic phosphate (U greater than p) as phosphate donor and 3-ribosyl-6-methyluracil (rmU) as phosphate acceptor. The rate of synthesis of UprmU is much higher than that of uridylyl-(3'-5')-uridine (UpU) in a control experiment under the same conditions with uridine as acceptor. The yields of UpU and UprmU were 20 and 17% respectively. The competitive hydrolysis of the initial U greater than p also proceeds faster when rmU is used as the acceptor. The relationship between the conformation of this nucleoside and its acceptor activity in the enzymatic synthesis of the internucleotide bond is discussed.     

Structural analysis and the effect of cyclo(His–Pro) dipeptide on neurotoxins—a dynamics and density functional theory study

The switching propensity and maximum probability of occurrence of the side chain imidazole group in the dipeptide cyclo(His–Pro) (CHP) were studied by applying molecular dynamics simulations and density functional theory. The atomistic behaviour of CHP with the neurotoxins glutamate (E) and paraquat (Pq) were also explored; E and Pq engage in hydrogen bond formation with the diketopiperazine (DKP) ring of the dipeptide, with which E shows a profound interaction, as confirmed further by NH and CO stretching vibrational frequencies. The effect of CHP was found to be greater on E than on Pq neurotoxin. A ring puckering study indicated a twist boat conformation for the six-membered DKP ring. Molecular electrostatic potential (MESP) mapping was also used to explore the hydrogen bond interactions prevailing between the neurotoxins and the DKP ring. The results of this study reveal that the DKP ring of the dipeptide CHP can be expected to play a significant role in reducing effects such as oxidative stress and cell death caused by neurotoxins.     

Hydrogen bonds in crystal structures of amino acids, peptides and related molecules

The results of a survey of 439 hydrogen bonds in 95 recently determined crystal structures of amino acids, peptides and related molecules suggest that the following generalizations hold true for linear (angle X-H---Y greater than 150 degrees) hydrogen bonds. (1) The charge on the acceptor group does not influence the length of a hydrogen bond. (2) For a given acceptor group, the hydrogen bond lengths increase in the order imidazolium N--H less than ammonium N-H less than guanidinium N-H; this order holds true for oxygen anion acceptor groups. Cl-ions and the uncharged oxygen of water molecules. (3) The uncharged imidazole N-H group forms shorter hydrogen than the amide N-H GROUP. (4) The carboxyl O-H groups form shorter hydrogen bonds than other hydroxyl groups. (5) The hydrogen bonds involving a halogen ion are longer than hydrogen bonds with other acceptors when corrected for their longer van der Walls radii. The observed differences between the lengths of hydrogen bonds formed by different donor and acceptor groups in amino acids and peptides, imply differences in the energetics of their formation.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Amide hydrogen exchange rates of peptides in H2O solution by 1H nuclear magnetic resonance transfer of solvent saturation method. Conformations of oxytocin and lysine vasopressin in aqueous solution.

The NH exchange rates in aqueous media of oxytocin and 8-lysine vasopressin (LVP) have been measured by using transfer of solvent saturation method. The data are consistent with a "highly motile" dynamic equilibrium between folded and highly solvated conformations. The highly-motility limit applies to the exchange of NH hydrogens of oxytocin and LVP. Folded structures are more prevalent in oxytocin than in LVP. Partial shielding is indicated for peptide hydrogens of Asn5 and perhaps also Cys6 of oxytocin and for Cys6 of LVP. It is tentatively proposed that the folded conformation of oxytocin in aqueous media may contain a parallel beta-structure in the tocinamide ring consisting of two hydrogen bonds: one between the Tyr2 C = O and Asn5 peptide NH as originally proposed for the preferred conformation of oxytocin in dimethyl sulfoxide (D. W. Urry and R. Walter), and the second between he Cys1 C = O and the Cys6 NH. In LVP the hydrogen bond between the Tyr2 C = O and Asn5 peptide NH appears to be absent. The acylic tripeptide sequences (-Pro-X-Gly-NH2) of both hormones appear to be predominantly solvated. The second-order rate constants for acid catalyzed exchange of the primary amide hydrogens of Gln4, Asn5, and Gly9 of oxytocin are consistently greater for the trans NH than for the corresponding cis NH. This observation can be rationalized in terms of mechanisms involving protonation of either the amide oxygen, or the amide nitrogen, but with limited rotation about the C - N bond.     

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Biological recognition of phosphate and sulfate

The biological recognition of trigonal pyramids has been studied in a statistical analysis of the Cambridge Structural Data Base. The preferential stereochemistry of pyramidal anion-Lewis acid interactions is easily described for the phosphonyl dianion (R-PO3(2-), as found in the phosphate group) and the sulfonyl monoanion (R-SO3-, as found in the sulfate group) by trans/gauche conformational terminology. Interactions between these pyramidal anions and metals generally prefer gauche geometry (as defined by the R-X = O-Mn+ torsion angle, X = P or S). Interactions between phosphonyls and hydrogen bond donors likewise display a preference for gauche orientation. Interactions between sulfonyls and hydrogen bond donors exhibit a preference for gauche stereochemistry and also cluster in eclipsed regions. These hydrogen bond motifs provide a greater understanding of the function of pyramidal anions in biological structure and function. For example, interactions of the sulfate monoanion are important for the binding of heparin, a sulfated glycosaminoglycan, to antithrombin III.     

Studies on hydrogen bonds. Part V--Hydrogen bonding in energy minimization studies of peptides

An energy term, representing the N-H...O type of hydrogen bond, which is a function of the hydrogen bond length (R) and angle (theta) has been introduced in an energy minimization program, taking into consideration its interpolation with the non-bonded energy for borderline values of R and theta. The details of the mathematical formulation of the derivatives of the hydrogen bond function as applicable to the energy minimization have been given. The minimization technique has been applied to hydrogen bonded two and three linked peptide units (gamma-turns and beta-turns), and having Gly, Ala and Pro side chains. Some of the conformational highlights of the resulting minimum energy conformations are a) the occurrence of the expected 4----1 hydrogen bond in all of the burn-turn tripeptide sequences and b) the presence of an additional 3----1 hydrogen bond in some of the type I and II tripeptides with the hydrogen bonding scheme in such type I beta-turns occurring in a bifurcated form. These and other conformational features have been discussed in the light of experimental evidence and theoretical predictions of other workers.     

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

Structure and conformation of peptides containing the sulfonamide junction. II. Synthesis and conformation of cyclo[-MeTau-Phe-DPro-]

By applying the method of amino-acyl incorporation to sulfonamido peptides, cyclo(-MeTau-Phe-DPro-) 3 has been synthesized in high yield starting from Z-MeTau-Phe-Pro-OH. The crystal structure and the molecular conformation of 3 have been determined. Crystals are orthorhombic, s.g. P2(1)2(1)2(1), with a = 5.454, b = 13.486, c = 24.025 A. The structure has been solved by direct methods and refined to R = 0.039 for 1974 reflections with I greater than 1.5 sigma (I). The 10-measured cyclopeptide adopts a backbone conformation in the crystals characterized by Phe-DPro and DPro-MeTau peptide bonds in trans and cis conformation, respectively. Both the peptide bonds deviate significantly from planarity and the corresponding [delta omega[ values are ca. 12 degrees. The sulfonamide SO2NH junction adopts a cisoidal conformation with a C alpha 1-S1-N2-C alpha 2 torsion angle of 70.8 degrees. 13C n.m.r. data show that the trans geometry at the Phe-DPro junction found in the crystals is retained in DMSO solution. The 10-membered ring of 3 is characterized by a pseudo mirror-plane passing through the Phe nitrogen and the DPro carbonylic carbon. The DPro ring adopts a half-chair conformation. The Phe side chain conformation corresponds to the statistically most favored g- rotamer (chi 1 = -68.6 degrees). The crystal packing is characterized by a weak intermolecular hydrogen bond between NH group and the MeTau O1' oxygen.     

Influence of the environment in the conformation of alpha-helices studied by protein database search and molecular dynamics simulations

The influence of the solvent on the main-chain conformation (phi and Psi dihedral angles) of alpha-helices has been studied by complementary approaches. A first approach consisted in surveying crystal structures of both soluble and membrane proteins. The residues of analysis were further classified as exposed to either the water (polar solvent) or the lipid (apolar solvent) environment or buried to the core of the protein (intermediate polarity). The statistical results show that the more polar the environment, the lower the value of phi(i) and the higher the value of Psi(i) are. The intrahelical hydrogen bond distance increases in water-exposed residues due to the additional hydrogen bond between the peptide carbonyl oxygen and the aqueous environment. A second approach involved nanosecond molecular dynamics simulations of poly-Ala alpha-helices in environments of different polarity: water to mimic hydrophilic environments that can form hydrogen bonds with the peptide carbonyl oxygen and methane to mimic hydrophobic environments without this hydrogen bond capabilities. These simulations reproduce similar effects in phi and Psi angles and intrahelical hydrogen bond distance and angle as observed in the protein survey analysis. The magnitude of the intrahelical hydrogen bond in the methane environment is stronger than in the water environment, suggesting that alpha-helices in membrane-embedded proteins are less flexible than in soluble proteins. There is a remarkable coincidence between the phi and Psi angles obtained in the analysis of residues exposed to the lipid in membrane proteins and the results from computer simulations in methane, which suggests that this simulation protocol properly mimic the lipidic cell membrane and reproduce several structural characteristics of membrane-embedded proteins. Finally, we have compared the phi and Psi torsional angles of Pro kinks in membrane protein crystal structures and in computer simulations.