Nonpolar Amino Acid Side Chains Research Articles

Desolvation of Peptide Bond by O to S Substitution Impacts Protein Stability

AbstractAmino acid side chains are key to fine‐tuning the microenvironment polarity in proteins composed of polar amide bonds. Here, we report that substituting an oxygen atom of the backbone amide bond with sulfur atom desolvates the thioamide bond, thereby increasing its lipophilicity. The impact of such local desolvation by O to S substitution in proteins was tested by synthesizing thioamidated variants of Pin1 WW domain. We observe that a thioamide acts in synergy with nonpolar amino acid side chains to reduce the microenvironment polarity and increase protein stability by more than 14 °C. Through favorable van der Waals and hydrogen bonding interactions, this single atom substitution significantly stabilizes proteins without altering the amino acid sequence and structure of the native protein.

Desolvation of Peptide Bond by O to S Substitution Impacts Protein Stability.

Amino acid side chains are key to fine-tuning the microenvironment polarity in proteins composed of polar amide bonds. Here, we report that substituting an oxygen atom of the backbone amide bond with sulfur atom desolvates the thioamide bond, thereby increasing its lipophilicity. The impact of such local desolvation by O to S substitution in proteins was tested by synthesizing thioamidated variants of Pin1 WW domain. We observe that a thioamide acts in synergy with nonpolar amino acid side chains to reduce the microenvironment polarity and increase protein stability by more than 14 °C. Through favorable van der Waals and hydrogen bonding interactions, this single atom substitution significantly stabilizes proteins without altering the amino acid sequence and structure of the native protein.

Apolar chemical environments compact unfolded RNAs and can promote folding

It is well documented that the structure, and thus function, of nucleic acids depends on the chemical environment surrounding them, which often includes potential proteinaceous binding partners. The nonpolar amino acid side chains of these proteins will invariably alter the polarity of the local chemical environment around the nucleic acid. However, we are only beginning to understand how environmental polarity generally influences the structural and energetic properties of RNA folding. Here, we use a series of aqueous-organic cosolvent mixtures to systematically modulate the solvent polarity around two different RNA folding constructs that can form either secondary or tertiary structural elements. Using single-molecule Förster resonance energy transfer spectroscopy to simultaneously monitor the structural and energetic properties of these RNAs, we show that the unfolded conformations of both model RNAs become more compact in apolar environments characterized by dielectric constants less than that of pure water. In the case of tertiary structure formation, this compaction also gives rise to more energetically favorable folding. We propose that these physical changes arise from an enhanced accumulation of counterions in the low dielectric environment surrounding the unfolded RNA.

Packing of apolar side chains enables accurate design of highly stable membrane proteins.

The features that stabilize the structures of membrane proteins remain poorly understood. Polar interactions contribute modestly, and the hydrophobic effect contributes little to the energetics of apolar side-chain packing in membranes. Disruption of steric packing can destabilize the native folds of membrane proteins, but is packing alone sufficient to drive folding in lipids? If so, then membrane proteins stabilized by this feature should be readily designed and structurally characterized-yet this has not been achieved. Through simulation of the natural protein phospholamban and redesign of variants, we define a steric packing code underlying its assembly. Synthetic membrane proteins designed using this code and stabilized entirely by apolar side chains conform to the intended fold. Although highly stable, the steric complementarity required for their folding is surprisingly stringent. Structural informatics shows that the designed packing motif recurs across the proteome, emphasizing a prominent role for precise apolar packing in membrane protein folding, stabilization, and evolution.

Precise packing for membrane proteins

Protein Design Although nonpolar amino acid side chains pack efficiently in membrane proteins, it has been difficult to determine how much this contributes to membrane protein stability. Designed membrane proteins have largely relied on other stabilizing interactions such as metal-ligand interactions and hydrogen bonds. Mravic et al. uncovered a steric packing code underlying the folding of the natural protein phospholamban, which they used to design stable membrane proteins with nonpolar interfaces. They suggest that packing of nonpolar residues plays a role in the folding and stability of many membrane proteins. Science , this issue p. [1418][1] [1]: /lookup/doi/10.1126/science.aav7541

Understanding wetting at nano scale: Atomistic simulation investigations

The wetting property of water is crucial in a variety of chemical, biological and technological systems. Finding the relevance between water and the structure of material surface is the key to understand the wettability of water and other implications such as those design nanostructured devices. In those work, we attempt to use the classical and the first-principles molecular dynamics simulations to explore the interactions between water and material surfaces. We have achieved a series of results, mainly including the following aspects: (1) Wettability of graphene and h-BN. (2) Relations between wettability and lattice constant. (3) Wetting of synthesized Bi2Se3. The water contact angle of the ideal Bi2Se3 surface is ~98.4°, and the edge of the terrace on its surface is extremely hydrophilic. (4) Wenzel to Cassie state transition. (5) Characterizing hydrophobicities of amino acid side chains. (6) Hydrophobicity and ion-selectivity. According to these examples, it is found that the molecular dynamics simulation is an extremely important tool to reveal the mechanism of wetting. In wettability of graphene and h-BN, we used Born-Oppenheim quantum molecular dynamics (QMD) simulations and investigated interfacial properties of water droplets on a graphene sheet or a monolayer BN sheet. The QMD simulations reveal that the contact angle of the graphene is 87°, while the contact angle of the monolayer BN sheet is 86°. So they all show weakly hydrophobic. Compared with nanodroplets of water in a supercooled state on the graphene, the results show that under the supercooled condition, water droplets exhibit an appreciably larger contact angle than under the ambient condition. In addition, there are also related introductions about evolution of the interfacial shape in dissolutive wetting and electrowetting. In the relations between wettability and lattice constant, the MD simulation presents a small uniform strain (±3%) applied to the lattice constant of a multilayer hydrophilic surface can generate a marked change in the wetting tendency. Meanwhile, when the lattice constant of a hydrophilic surface matches the projected oxygen-oxygen distance of bulk water to the surface, a contact-angle minimum is resulted. They surmised that the structure of the first water layer next to the surface can strongly affect the contact angle of water droplets. For vdW layered materials surface and nanostructured surfaces by design, the MD simulations show that a modified Wenzel model can proposed for describing the wetting behavior of van der Waals layered materials with topographic surfaces and the water contact angle of the ideal Bi2Se3 surface is ~98.4°. The Cassie state may translate into Wenzel state via precisely designed trapezoidal nanostructures. Two key parameters—The base angle of the trapezoids and the intrinsic contact angle—can be controlled the transition. For example, there will be three regimes for a given base angle. For characterizing hydrophobicity of amino acid side chains in a protein environment, the hydrophobicity of 20 types of amino acids has been studied by measuring the CA ( θ ) of a water nanodroplet. The results show that all nonpolar amino acid side chains are hydrophobic and the difference in θ among the nonpolar amino acid side chains is very small. Finally, we introduced the mechanism of selective ion transport in hydrophobic subnanometer channels, intercalation and diffusion of lithium inos in a carbon nanotube bundle and a series of related issues.

Peptide backbone effect on hydration free energies of amino acid side chains.

We have studied the hydrophobicity of amino acid side chains by computing conditional solvation free energies that account for effects of the peptide backbone on the side chains' solvent environment. The free energies reported herein correspond to a gas-liquid transfer process, which mimics solvation of the side chain under the condition that the backbone has been solvated already, and have been obtained on the basis of free energy calculations with empirical force field models. We find that the peptide backbone strongly impacts the solvation of nonpolar side chains, while its effect on the polar side chains is less pronounced. The results indicate that, in the presence of the short peptide backbone, nonpolar amino acid side chains are less hydrophobic than what is expected based on small molecule (analogue) solvation data.

Antibiotic Selection by the Promiscuous Aminoglycoside Acetyltransferase-(3)-IIIb Is Thermodynamically Achieved through the Control of Solvent Rearrangement

The results presented here show the first known observation of opposite signs of change in heat capacity (ΔC(p)) of two structurally similar ligands binding to the same protein site. Neomycin and paromomycin are aminoglycoside antibiotics that are substrates for the resistance-conferring enzyme, the aminoglycoside acetyltransferase-(3)-IIIb (AAC). These antibiotics are identical to one another except at the 6' position where neomycin has an amine and paromomycin has a hydroxyl. The opposite trends in ΔC(p) of binding of these two drugs to AAC suggest a differential exposure of nonpolar amino acid side chains. Nuclear magnetic resonance experiments further demonstrate significantly different changes in AAC upon interaction with neomycin and paromomycin. Experiments in H(2)O and D(2)O reveal the first observed temperature dependence of solvent and vibrational contributions to ΔC(p). Coenzyme A significantly influences these effects. Together, the data suggest that AAC exploits solvent properties to facilitate favorable thermodynamic selection of antibiotics.

Cover Picture: Learning from Directed Evolution: Thermus aquaticus DNA Polymerase Mutants with Translesion Synthesis Activity (ChemBioChem 10/2011)

The cover picture shows a crystal structure of KenTaq DNA polymerase bypassing an abasic site analogue. Abasic sites are the most frequent lesions to arise under physiological conditions and represent a strong block for replicative DNA polymerases. However, nature offers a vast arsenal of specialized DNA polymerases that have evolved for translesion bypass. On p. 1574 ff., A. Marx et al. show how, starting from DNA polymerase that is incompetent in translesion synthesis, they substituted two nonpolar amino acid side chains with two cationic side chains to give a DNA polymerase mutant with enhanced translesion synthesis capability. The mutant shows expanded positively charged surface potential areas, which are also found in naturally evolved translesion DNA polymerases. The results underscore the impact of charge on the proficiency of naturally evolved translesion DNA polymerases.

Backbone-Driven Collapse in Unfolded Protein Chains

Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.

Purification of Large “Troublesome” Polypeptides by RP-HPLC

INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troublesome to handle because of their low solubility or the presence of secondary structural elements that favor supramolecular self-self assembly. Included in this group are polypeptides with amphipathic α-helical or β-sheet structures with extensive runs of nonpolar (hydrophobic) amino acid side chains or proline-rich sequences. RP-HPLC provides one avenue to purify such troublesome examples provided certain steps and precautions are taken. (Related procedures are equally germane to polypeptides that have been lipidated or subjected to chemical modifications with nonpolar moieties, as well as to some core cyanogen bromide fragments of large proteins.) RP-HPLC methods not only enable concomitant desalting, removal of additives that aid dissolution of the polypeptide or protein, but also permit resolution and maintenance of a reasonably high concentration of the solutes, due to the presence of the (often) low-pH, aquo-organic solvent conditions.

Mercedes Benz model of neutral amino-acid side chains

We present a 2-d coarse-grain model of polar and non-polar amino-acid side chains, and investigate their thermodynamic and structural properties in solution. The molecules interact via a Lennard-Jones potential and a local treatment of the hydrogen bond energy. Such a form allows very fast highly converged calculations. Estimates of the Gibbs free energy of hydration (GFEH) of the solutes show good qualitative agreement with experiments. We find moreover that contributions to the GFEH are linked to the perturbations of the solvent hydration shell structure. For large hydrophobic solutes, unfavorable GFEH results mainly from an increase of the water–water energy contribution, while for hydrophilic solutes the increase in solvent energy is compensated by the solute–solvent energy. The model appears therefore to capture the main features of non-charged amino-acids hydration.

A pulsed field gradient NMR diffusion investigation of enkephalin peptide‐sodium dodecyl sulfate micelle association

Pulsed field gradient NMR (PFG-NMR) diffusion experiments were used to investigate the binding of leucine and methionine enkephalin peptides to anionic sodium dodecyl sulfate (SDS) micelles. The study was undertaken to investigate the mechanism of interaction between enkephalin peptides and SDS micelles and to determine if NMR-derived association constants, K(eq), can predict the elution order in electrokinetic chromatography (EKC). In EKC, peptides are separated on the basis of their interactions with micelles. The Leu-enkephalin peptide-micelle association constant increased from 130 +/- 8 to 1459 +/- 57 and 1744 +/- 64 M(-1), respectively, when an Arg or Lys was added to the C-terminus. The association constant of Leu-enkephalinamide was approximately equal to that of Leu-enkephalin-Arg. Substitution of Phe4 with a Trp or Gly2 with an Ala in the Leu-enkephalin peptides also increased the micelle binding affinity. These results confirm that the interaction of Leu-enkephalin peptides with SDS micelles is largely electrostatic and that the non-polar amino acid side chains interact with the hydrophobic micelle core. The peptide-micelle association constants for the cationic Met-enkephalin peptides were also greater than their zwitterionic counterparts. For example, the Met-enkephalin K(eq) value was 162 +/- 9 M(-1), while the association constants for Met-enkephalin-Arg, Met-enkephalin-Lys, and Met-enkephalinamide were, respectively, 674 +/- 31, 426 +/- 23, and 453 +/- 27 M(-1). In both Met-enkephalin and Met-enkephalinamide, replacing Gly2 with an Ala did not significantly increase the association constant. These results confirm that with the Met-enkephalin peptides, there was little or no interaction of the amino acid side chains with the micelle core. For both the Leu-enkephalin and Met-enkephalin peptides, the association constants were consistent with EKC results, in that the peptides with smaller K(eq) values were found to elute before those with larger association constants.

Hydration properties and potentials of mean force of nonpolar amino acid residues in water: A pertubation theoretic approach

We use a perturbation approach to calculate the solute-solvent and solute-solute pair correlations for infinitely dilute solutions of nonpolar amino acid side chains in water. In particular, from the solute-solute correlations we derive potentials of mean force for all the distances between different residues of amino acids. Comparison with molecular dynamics simulations performed using GROMOS package shows that the goodness of the approximation varies with the amino acid considered. We discuss in terms of hydrophobicity the different effect that the inclusion of the attractions causes on the solute-water and solute-solute correlations.

Molecular Simulation Study of the Potentials of Mean Force for the Interactions between Models of Like-Charged and between Charged and Nonpolar Amino Acid Side Chains in Water

The purpose of this study was to determine the potentials of mean force (PMF) of the interactions between models of like-charged and between models of charged and nonpolar amino acid side chains in water to design improved side chain−side chain interaction potentials for our united residue UNRES force field for protein-structure prediction. Restrained molecular dynamics with the AMBER force field, the TIP3P model of water, and the Ewald summation were used to carry out simulations, and the weighted histogram analysis method (WHAM) was used to calculate the PMFs as functions of solute−solute distances. The following types of systems were considered to model the interactions between like-charged side chains and the interactions between charged and nonpolar side chains: (i) a pair of positively charged ions (potassium, ammonium, and guanidine, respectively); (ii) a pair of negatively charged ions (chloride and acetate, respectively), and (iii) pairs of methane with potassium, ammonium, and chloride ions, re...

Hydrophobic Residue Contributions to Sequence-Specific DNA Binding by the Bovine Papillomavirus Helicase E1

Previously, mutational analyses of the DNA binding domain of the bovine papillomavirus E1 protein (E1DBD) identified several hydrophobic residues that are critical for DNA binding activity (M. West, D. Flanery, K. Woytek, D. Rangasamy, and V. G. Wilson, 2001, J. Virol. 75, 11948–11960). Hydrophobic interactions of nonpolar amino acid side chains can contribute to the function of DNA binding proteins through both conformational effects and direct interaction with nucleotides. To further investigate the role of hydrophobic residues in E1DBD function, a more extensive site-directed mutational analysis of hydrophobic amino acids was conducted. Alanine substitutions were made at residues V196, F197, F217, F, 237, V246, L249, and F276, and the mutants were tested for DNA binding activity in vitro and in vivo. The E1 F237A and F276A mutants were completely defective for site-specific DNA binding, while the other mutants retained partial to full wild-type binding activity. Consistent with their DNA binding defect, the F237A and F276A mutants were severely impaired for the ability to support transient in vivo replication of an origin plasmid. Combined with our previous study, five critical hydrophobic residues have been identified: F175, V193, F237, V246, and F276. These five residues localize to two internal clusters in the E1DBD structure designated hydrophobic clusters A (HCA; includes F175, V193, and F276) and B (HCB; includes F237 and V246). Amino acid side chains from residues in HCA and HCB have little surface accessibility and it is unlikely that they are involved in direct contact with DNA. HCA is distal to the DNA binding surface and presumably contributes to global conformational organization of the E1DBD. HCB is positioned beneath the DNA contact surface and we propose that it serves as an anchor or platform device to stabilize the DNA-binding element. A comparable hydrophobic cluster is present in the corresponding position in the T antigen DBD and likely serves a similar function.

Effect of Amino Acid Sequence Change on Peptide−Membrane Interaction

The RGD region of the (110−121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus, which is described to be responsible for a high immunoresponse, was changed for RGE and RKD amino acids in order to analyze the change effect on the physicochemical properties of the peptide. Peptides were synthesized by solid-phase synthesis and characterized by amino acid analysis, analytical high-performance liquid chromatography, and electrospray mass spectrometry. The peptides had surface activity concentration dependent, formed stable monolayers at the air/water interface, and were able to insert into lipid monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol, phosphatydic acid, l-α-phosphatidyl-l-serine, stearylamine, sphyngomyelin, and liver extract. Differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of DPPC and the three peptides. DSC data showed a complete abolition of DPPC pretransition and significant broadening of the main phase transition with increasing amounts of peptide. These results are indicative of an interfacial location of the peptides and with some penetration of the nonpolar amino acid side chains into the hydrocarbon chain region closer to the polar/nonpolar interface. Finally, fluorescence spectroscopy confirmed the preferential interaction of the peptides with the liquid crystalline state of the bilayer with a contribution of both hydrophobic and electrostatic forces.

Binding thermodynamics of the transition state analogue coformycin and of the ground state analogue 1-deazaadenosine to bovine adenosine deaminase.

Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermodynamically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are -4.7 +/- 0.8 kJ/mole-K and -1.2 +/- 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect. Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.

Infinite dilution activity coefficients for benzene and toluene in water and in aqueous solutions of the protein denaturants urea and guanidine hydrochloride

Experimental infinite dilution activity coefficientsγ1∞ for benzene and toluene in water and in aqueous solutions of two protein denaturants, urea and guanidine hydrochloride, are reported. The quantities were measured in the temperature range (273 to 323) K. Four denaturant concentrations were used from (0.5 to 5.0)mol · dm−3, covering the range where proteins undergo chemical denaturation. For both solutes in water, from the freezing temperature of water, γ1∞rises with increasing temperature, passes through a maximum close to room temperature, and then continuously decreases with increasing temperature. Based on the results reported here and selected literature values, recommended correlations ofγ1∞ (T) for benzene and toluene in water were established. They clearly show that from the freezing temperature to the normal boiling temperature of water, the origin of the hydrophobicity of the non-polar solutes changes from being entropic to enthalpic in nature. The presence of a denaturant (modifier) produces a significant decrease of γ1∞values, their maximum being displaced towards lower temperatures. The effect is more pronounced for guanidine hydrochloride, which acts as a better solubilizing agent than urea. The solute in (water + modifier)γ1∞data were analysed by a classical thermodynamic scheme employing solution and transfer {water to (water + modifier)} quantities. The accuracy of the data allowed the derivation of enthalpies, entropies, and heat capacities and a detailed discussion of the several observed trends. A remarkable feature is that there is only a narrow temperature interval where both the enthalpic and entropic contributions to the Gibbs energy are favourable to solute transfer. At lower and higher temperatures, the enthalpic and entropic components compete, solute transfer being favoured by entropy or by enthalpy, respectively. Extrapolation of this behaviour suggests that the effect of the modifier on the solubility of hydrophobic solutes should eventually invert at both lower and higher temperatures. The data presented here might be used to understand better, through the application of different models, the exposure of non-polar amino acid side chains from the protein interior to the aqueous environment, which characterizes protein denaturation.

Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure.

Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.