Analysis of the structural and dynamic properties of human N-terminal domain of apolipoprotein E by molecular dynamics simulations

Whereas the lipid-free N-terminal domain of apolipoprotein E (apoE-NT) adopts a four-helix bundle, the lipid-bound form is believed to undergo a large conformational change likely to be characterized by the opening of the bundle. ApoE-NT in a water/alcohol mixture was also shown to experience conformational changes exhibiting similarities with those induced upon lipid binding. The structure and dynamics of apoE-NT have been here investigated by analyzing 40 ns and 60 ns molecular dynamics simulations performed in water and in a water/propanol mixture, respectively. The overall structural properties show alterations of the tertiary structure of apoE-NT in the water/alcohol system in agreement with those observed experimentally. In contrast, in the water simulation, the sampled conformations remain closer to the crystal structure that served as a starting point for both simulations. Interestingly, several propanol molecules are seen to penetrate two hydrophobic regions of the bundle interior. One of these regions is enclosed in part by the short helix (H1') connecting helices 1 and 2 of the bundle which has been experimentally shown to be important for modulating lipid binding activity of apoE-NT. Principal component analysis of the water/propanol trajectory confirms that the region including H1' is the locus of the largest motion. Another region involves the loop connecting helix 2 and helix 3 which has been hypothesized to play the role of a hinge in the opening of the bundle.     

Interactions of peptide mimics of hyaluronic acid with the receptor for hyaluronan mediated motility (RHAMM)

Summary Using the hyaluronic acid (HA) binding region of the receptor for hyaluronan-mediated motility (RHAMM) as a model, a molecular perspective for peptide mimicry of the natural ligand was established by comparing the interaction sites of HA and unnatural peptide–ligands to RHAMM. This was accomplished by obtaining a series of octapeptide–ligands through screening experiments that bound to the HA binding domains of RHAMM (amino acids 517–576) and could be displaced by HA. These molecules were computationally docked onto a three-dimensional NMR based model of RHAMM. The NMR model showed that RHAMM(517–576) was a set of three helices, two of which contained the HA binding domains (HABDs) flanking a central groove. The structure was stabilized by hydrophobic interactions from four pairs of Val and Ile side chains extending into the groove. The presence of solvent exposed, positively charged side chains spaced 11Å apart matched the spacing of negative charges on HA. Docking experiments using flexible natural and artificial ligands demonstrated that HA and peptide–mimetics preferentially bound to the second helix that contains HABD-2. Three salt bridges between HA carboxylates and Lys548, Lys553 and Lys560 and two hydrophobic interactions involving Val538 and Val559 were predicted to stabilize the RHAMM-HA complex. The high affinity peptides and HA utilized the same charged residues, with additional contacts to other basic residues. However, hydrophobic contacts do not contribute to affinity for peptide ligand-RHAMM complexes. These results offer insight into how selectivity is achieved in the binding of HA to RHAMM, and how peptide competitors may compete for binding with HA on a single hyaladherin.     

Putative One‐Pot Prebiotic Polypeptides with Ribonucleolytic Activity

KIA7, a peptide with a highly restricted set of amino acids (Lys, Ile, Ala, Gly and Tyr), adopts a specifically folded structure. Some amino acids, including Lys, Ile, Ala, Gly and His, form under the same putative prebiotic conditions, whereas different conditions are needed for producing Tyr, Phe and Trp. Herein, we report the 3D structure and conformational stability of the peptide KIA7H, which is composed of only Lys, Ile, Ala, Gly and His. When the imidazole group is neutral, this 20‐mer peptide adopts a four‐helix bundle with a specifically packed hydrophobic core. Therefore, one‐pot prebiotic proteins with well‐defined structures might have arisen early in chemical evolution. The Trp variant, KIA7W, was also studied. It adopts a 3D structure similar to that of KIA7H and its previously studied Tyr and Phe variants, but is remarkably more stable. When tested for ribonucleolytic activity, KIA7H, KIA7W and even short, unstructured peptides rich in His and Lys, in combination with Mg⁺⁺, Mn⁺⁺ or Ni⁺⁺ (but not Cu⁺⁺, Zn⁺⁺ or EDTA) specifically cleave the single‐stranded region in an RNA stem–loop. This suggests that prebiotic peptide–divalent cation complexes with ribonucleolytic activity might have co‐inhabited the RNA world.     

BUNDLE: A program for building the transmembrane domains of G-protein-coupled receptors

The only information available at present about the structural features of G-protein-coupled receptors (GPCRs) comes from low resolution electron density maps of rhodopsin obtained from electron microscopy studies on 2D crystals. Despite their low resolution, maps can be used to extract information about transmembrane helix relative positions and their tilt. This information, together with a reliable algorithm to assess the residues involved in each of the membrane spanning regions, can be used to construct a 3D model of the transmembrane domains of rhodopsin at atomic resolution. In the present work, we describe an automated procedure applicable to generate such a model and, in general, to construct a 3D model of any given GPCR with the only assumption that it adopts the same helix arrangement as in rhodopsin. The present approach avoids uncertainties associated with other procedures available for constructing models of GPCRs based on a template, since sequence identity among GPCRs of different families in most of the cases is not significant. The steps involved in the construction of the model are: (i) locate the centers of the helices according to the low-resolution electron density map; (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map; (iii) define a local coordinate system for each of the helices; (iv) bring them together in an antiparallel orientation; (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction of the bisector formed between three consecutive helices in the bundle; (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt; and (vii) translate each helix to its center deduced from the projection map.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Boron cluster-based development of potent nonsecosteroidal vitamin D receptor ligands: direct observation of hydrophobic interaction between protein surface and carborane

We report here the design and synthesis of a novel vitamin D receptor (VDR) agonist whose hydrophobic core structure is p-carborane (1,12-dicarba-closo-dodecaborane, an icosahedral carbon-containing boron cluster having remarkable thermal and chemical stability and a characteristically hydrophobic B-H surface). This carborane-based VDR ligand exhibited moderate vitamin D activity, comparable to that of the natural hormone, despite its simple and flexible structure. X-ray structure analysis provided direct evidence that the carborane cage binds to the hydrophobic surface of the ligand-binding pocket of the receptor, promoting transition to the active conformation. These results indicate that the spherical B-H surface of carborane can function efficiently as a hydrophobic anchor in binding to the receptor surface, thereby allowing induced fitting of the three essential hydroxyl groups on the alkyl chains to the appropriate positions for interaction with the VDR binding site, despite the entropic disadvantage of the flexible structure. We suggest that carborane structure is a promising option in the design of novel drug candidates.     

A general procedure for building the transmembrane domains of G‐protein coupled receptors

The only results available at present about the structural features of G‐protein coupled receptors are the low resolution electron projection maps obtained from microscopy studies carried out on two‐dimensional crystals of rhodopsin. These studies support previous suggestions that these integral proteins are constituted by seven transmembrane domains. The low resolution electron density map of rhodopsin can be used to extract information about helix relative positions and tilt. This information, together with a reliable procedure to assess the residues involved in each of the transmembrane regions, can be used to construct a model of rhodopsin at atomic resolution. We have developed an algorithm that can be used to generate such a model in a completely automated fashion. The steps involved are: (i) locate the centers of the helices according to the low resolution electron density map; (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map; (iii) define a local coordinate system for each of the helices; (iv) bring them together in an antiparallel orientation; (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction as the bisector formed between three consecutive helices in the bundle; (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt; (vii) translate each of the helix to its center deduced from the projection map. A major advantage of the procedure presented is its generality and consequently can be used to obtain a model of any G‐protein coupled receptor with the only assumption that the shape of the bundle is the same as found in rhodopsin. This avoids uncertainties found in other procedures that construct models of G‐protein coupled receptors based on sequence homology using rhodopsin as template. This revised version was published online in July 2006 with corrections to the Cover Date.     

Experimental resolution of early steps in protein folding: testing molecular dynamics simulations

Time-resolved Tyr fluorescence spectroscopy coupled with a laser-induced temperature-jump (T-jump) was employed to follow the folding relaxation dynamics of the B-domain of Staphylococcal protein A. The single Tyr is located in helix 1 (H1) and is a sensitive probe of the structure of this helix and the overall helical bundle structure. The results from this study were compared to those from a complementary infrared T-jump study on this protein [Vu, D. M.; Myers, J. K.; Oas, T. G.; Dyer, R. B. Biochemistry 2004, 43, 3582]. Both methods detect a microsecond process that follows the cooperative relaxation of the helical bundle core. However, a fast process (10-7 s) that follows the relaxation of the individual helices was observed only with the infrared probe. Thus, fast formation of H1 is not observed, but rather H1 forms in the microsecond phase, concomitantly with the docking to (and stabilization by) the other two helices to form the helical bundle structure. This observation validates the results of several previous molecular dynamics simulations that predict H1 formation only in the final assembly of the helix bundle.     

Direct and high resolution characterization of cytochrome c equilibrium folding

Protein folding has emerged as a central problem in biophysics, and the equilibrium folding mechanism of cytochrome c (cyt c) has served as a model system. Unfortunately, the detailed characterization of both the folding process and of any intermediate that might be populated has been limited by the low structural and/or temporal resolution of the available techniques. Here, we report the use of a recently developed technique to study folding that is based on the site-selective incorporation of carbon-deuterium (C-D) bonds and their characterization by IR spectroscopy. Specifically, we synthesize and characterize the protein with deuterated residues spread throughout four structural motifs: (d3)Leu68 in the 60's helix, (d8)Lys72 and (d8)Lys73 in the 70's helix, (d8)Lys79, (d3)Met80, and (d3)Ala83 in the D-loop, and (d3)Leu94, (d3)Leu98, and (d3)Ala101 in the C-terminal helix. The data reveal correlated behavior of the residues within each structural motif, as well as between the residues of the 60's and C-terminal helices and between residues of the 70's helix and D-loop. Residues of the 70's helix and the D-loop are more stable than those within the 60's and C-terminal helices, although the former are more sensitive to added denaturant. The data also suggest that the hydrophobicity of the heme cofactor plays a central role in folding. These results contrast with those from previous H/D exchange studies and suggest that the low denaturant fluctuations observed in the H/D exchange experiments are not similar to those through which the protein actually unfolds. The inherently fast time scale of IR also allows us to characterize the folding intermediate, long thought to be present, but which has proven difficult to characterize by other techniques.     

Functionally Important Aromatic-Aromatic and Sulfur-π Interactions in the D2 Dopamine Receptor

The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46, and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51, show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic-aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur-π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.     

Structural effects on the formation of proton and alkali metal ion adducts of apolar,neutral peptides: electrospray ionization mass spectrometry and Ab initio theoretical studies

Apolar, neutral peptides have been shown to ionize extremely well under the conditions used for electrospray ionization mass spectrometry (ESIMS). Peptides for which the conformations have been independently determined in solution and in crystals have been examined by ESIMS. Studies of peptide helices ranging from 7 to 18 residues reveal that shorter helices yield exclusively singly charged ions, while in larger helices multiply charged species are detectable. Multiple sites for protonation/metallation are introduced in the helix by proline insertion or by changing the chirality in the residue. The preferred site of cation binding to helices may be the C-terminus end, where three free CO groups are available for chelation. Ab initio and DFT calculations at several levels have been carried out for the binding of H+, Li+, Na+, and K+ to CHO-(Gly)3)-OMe. The results reveal that metallation in helices is favoured by chelation to carbonyl groups at the C-terminus, while protonation involved two carbonyl groups and thus favour a 10-membered cyclic hydrogen-bonded structure. In -strands, metallation/protonation occurs at isolated carbonyl groups. Collision induced fragmentation of hydrophobic peptides under ESI conditions reveals that helix fragmentation occurs predominantly from the C-terminus, while in -hairpins cleavage occurs simultaneously at multiple sites.     

Binding of the general anesthetics chloroform and 2,2,2-trichloroethanol to the hydrophobic core of a four-alpha-helix bundle proteins

The structural features of general anesthetic binding sites on proteins are being examined using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work suggested that halothane binding to the four-alpha-helix bundle was improved by (1) introducing a cavity into the hydrophobic core and (2) substituting a methionine side-chain in place of an alpha-helical heptad e position leucine. In this study, the ability of the general anesthetics chloroform and 2,2,2-trichloroethanol to bind to the hydrophobic core of the four-alpha-helix bundle (Aalpha2-L38M)2 is explored. The halogenated alkane chloroform binds with a dissociation constant (Kd) = 1.4 +/- 0.2 mM, whereas 2,2,2-trichloroethanol binds with a Kd = 19.5 +/- 1.2 mM. The affinity of both general anesthetics for the hydrophobic core of the four-alpha-helix bundle approximates their whole animal effective concentration in 50% of test subjects' (EC50) values, as shown previously for halothane. Tryptophan phosphorescence decay rates at 77 K are accelerated by a factor of 4.5 by both bound halothane and chloroform, indicating that the heavy-atom effect is responsible for a portion of the observed fluorescence quenching. Because heavy-atom effects are operative only at short distances, the findings indicate that these general anesthetics are binding in the vicinity of the indole rings of W15 in the hydrophobic core of the four-alpha-helix bundle scaffold. The results indicate that chloroform, halothane and 2,2,2-trichloroethanol may occupy the same sites on protein targets.     

Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G‐protein‐coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double‐cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G‐protein‐coupled Y₂ receptor (Y₂R). Solid‐state NMR measurements of specific isotope‐labeled NPY in complex with in vitro folded Y₂R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C‐terminal α‐helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T³² to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y₂R.     

Broad distribution of energetically important contacts across an extended protein interface

Infection of cells by HIV depends upon profound structural rearrangements within the trimeric viral protein gp41. Critical to this process is the formation of a six-helix bundle in which a set of three N-terminal heptad repeat (NHR) helices assemble to form a core displaying long grooves that provide docking sites for three C-terminal heptad repeat (CHR) helices. We report experiments designed to discriminate between two alternative hypotheses regarding the source of affinity between individual CHR helices and the complementary groove: (1) affinity is dominated by interactions of a small cluster of side chains at one end of the CHR helix; or (2) affinity depends upon interactions distributed across the long CHR helix. We have employed two complementary experimental designs, and results from both favor the latter hypothesis.     

Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions

We show that a single internal polar interaction per helix is sufficient to engender structural specificity in that helix in helical bundle proteins. Furthermore, we use histidine-binding cofactors of different shapes which bind directly into the core, demonstrating that this structural specificity is not the result of a prescribed complimentary, "knobs in holes" core packing. We show that we can switch structural specificity of individual helices on and off by ligating cofactors, singly and in pairs, which bind either one or two histidine ligands. To our knowledge, this is the first demonstration of such extensive manipulation of protein structure by ligand binding, an important result of general interest to those working with self-assembled molecular systems. Finally, as these proteins were designed without the use of computational modeling, we not only demonstrate that designing a uniquely structured cofactor binding protein is not as difficult as is generally believed, we have determined why this is so: hydrophobic core complementarity, which is very difficult to design, is not necessary. Instead, a much simpler design process entails the creation of core polar interactions which themselves can drive conformational specificity.     

De Novo Design,Synthesis and Characterisation of MP3, A New Catalytic Four‐Helix Bundle Hemeprotein

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four‐helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix–loop–helix/heme/helix–loop–helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP‐like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix‐forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe^III–MP3 possesses peroxidase‐like activity comparable to R38A–hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.     

A sequence and structural study of transmembrane helices

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the -branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline, residue types normally associated with `helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.     

Alamethicin channels in a membrane: molecular dynamics simulations

Alamethicin (Alm) is a 20 residue peptide which forms a kinked alpha-helix in membrane and membrane-mimetic environments. Ion channels formed by intramembraneous aggregates of Alm are thought to be formed by bundles of approximately parallel Alm helices surrounding a central bilayer pore. Different channel conductance levels correspond to different numbers of helices per bundle, ranging from N = 5 to N > 8. Calculation of the predicted pKA values of the ring of Glu18 sidechains at the C-terminal mouth of the pore suggests that at neutral pH most or all of these sidechains will remain protonated. Nanosecond molecular dynamics (MD) simulations of N = 5, 6, 7 and 8 bundles of Alm helices in a POPC bilayer have been run, corresponding to a total simulation time of 4 ns. These simulations explore the stability and conformational dynamics of these helix bundle channels when embedded in a full phospholipid bilayer in an aqueous environment. The structural and dynamic properties of water in these model channels are examined. As in earlier in vacuo simulations (J. Breed, R. Sankararamakrishnan, I. D. Kerr and M. S. P. Sansom, Biophys. J., 1996, 70, 1643) the dipole moments of water molecules within the pores are aligned antiparallel to the helix dipoles. This helps to contribute to the stability of the helix bundles.     

Reversed-phase liquid chromatography as a tool in the determination of the hydrophilicity/hydrophobicity of amino acid side-chains at a ligand-receptor interface in the presence of different aqueous environments. I. Effect of varying receptor hydrophobicity

We have developed further a chromatographic model for studying the hydrophobic interactions which characterize the way a ligand binds to its receptor. This model is based on observing the retention behaviour of de novo designed model 18-residue amphipathic alpha-helical peptides (representing the hydrophobic binding domain of a ligand) on reversed-phase packings by varying hydrophobicity (representing a receptor protein with a hydrophobic binding pocket). Mutants of the "native" peptide ligand (which contains seven Leu residues in its non-polar face) were designed by replacing one residue in the center of the extremely non-polar face of the amphipathic alpha-helix. Through reversed-phase liquid chromatography of these peptides at pH 2.0 on cyano and C18 columns, we have demonstrated how an increase in receptor hydrophobicity (represented by an increase in column stationary phase hydrophobicity; cyano --> C18) significantly enhances hydrophilicity of polar amino acid side-chains at the ligand-receptor interface while moderately enhancing the hydrophobicity of non-polar side-chains. The addition of salt (100 mM sodium perchlorate) to the aqueous environment surrounding the binding site of receptor and ligand was also shown to have a profound effect on side-chain hydrophilicity/hydrophobicity in the binding interface. This effect was particularly dramatic for the positively charged side-chains Arg, Lys and His, whose significant enhancement of hydrophobicity in the presence of the cyano column contrasted with their increase in hydrophilicity in the presence of the considerably more hydrophobic C18 stationary phase. Our results have major implications to understanding the influence of hydrophobic and aqueous environment on hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.