Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).     

Extension of the fragment method to calculate amino acid zwitterion and side chain partition coefficients

The fragment method of calculating partition coefficients (P) has been extended to include the common amino acids (AAs). The results indicate that polar and charged side chains influence the hydrophobicity of atoms in the side chain in a predictable manner. Field effects, as evidenced through polar proximity factors and bond factors, need to be considered for accurate estimation of transfer phenomena. The calculated log P and delta G degree ' values of the 20 AAs agree well with the observed values. Pro calculates to be more hydrophilic than the observed log P. Hydrophobicity scales for peptide side chain residues are compared and evaluated in terms of suitability. Calculated pi values for nonpolar side chain residues agree well with the observed values; calculated values for uncharged polar side chain residues deviate by about 0.6 log units except for Gln and Cys; and polar side chain residues with charged side chains calculate as too hydrophilic. Reasons for the differences are explored. We also suggest that tightly bound water to polar moieties in amino acids and peptides may be transferred into the octanol phase during partitioning experiments. A quantitative methodology is presented which characterizes the thermodynamic partitioning of groups and individual atoms in amino acids and proteins.     

Nonlinear feedforward control of bioreactors with input multiplicities

A steady-state nonlinear feedforward controller (FFC) for measurable disturbances is designed for a continuous bioreactor, which is represented by Hammerstein type nonlinear model wherein the nonlinearity is a polynomial with input multiplicities. The manipulated variable is the feed substrate concentration (S_f) and the disturbance variable is the dilution rate (D). The productivity (Q=DP) is considered as the controlled variable. The desired value of Q=3.73 gives two values of feed substrate concentration. The nonlinearity in the gain is considered for relating output to the manipulated variable and separately for the relation between output to disturbance variable. The FFC is also designed for the overall linearized system. The performance of the FFC is evaluated on the nonlinear differential equation model. The FFC is also designed for the model based on a single nonlinear steady-state equation containing both D and S_f. This nonlinear FFC gives the best performance. The nonlinear FFC is also designed by using only linear gain for the disturbance and nonlinear gain for the manipulated variable. Similarly, nonlinear FFC is also designed by using linear gain for the manipulated variable and the nonlinear gain for the disturbance variable. The performances of these FFC schemes are compared.     

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.     

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.     

Lipophilicity of amino acids

Summary The lipophilicity (or hydrophobicity) of amino acids is an important property relevant for protein folding and therefore of great interest in protein engineering. For peptides or peptidomimetics of potential therapeutic interest, lipophilicity is related to absorption and distribution, and thus indirectly relates to their bioactivity. A rationalization of peptide lipophilicity requires basic knowledge of the lipophilicity of the constituting amino acids. In the present contribution we will review methods to measure or calculate the lipophilicities of amino acids, including unusual amino acids, and we will make a comparison between various lipophilicity scales.     

An evaluation of the uniform stress hypothesis based on stem geometry in selected North American conifers

The uniform stress hypothesis of stem formation was evaluated by comparing stem taper of Abies balsamea, Abies lasiocarpa, Picea rubens, Pinus contorta, Pinus elliottii, Pinus palustris, Pinus ponderosa, Pinus taeda, and Pseudotsuga menziesii to the taper expected if stems develop to uniformly distribute bending stress. The comparison was conducted by regressing stem diameter at height h (D_h) against bending moment at h (M_h) using the model D_h=J (M_h)^' where J and ' are fitted coefficients, and testing for '=0.333, the hypothesized value. Twelve curves were fitted with the model. Seven of the fitted values of ' were significantly different from 0.333, but eight of the values were within ᆞ% of 0.333 and eleven values were within ᆣ% of 0.333. Where the fitted value of ' was >15% of 0.333, residuals were biased with height. Fit by relative height, values of ' were within ᆞ% of 0.333 for large portions of these stems. While most of the fitted values of ' support the uniform-stress hypothesis, the values of ' for Pseudotsuga menziesii trees clearly did not. Many of the fitted values of J were inversely related to the modulus of elasticity (E) of green wood reported for these species. With the exception of Pseudotsuga menziesii, growing conditions appeared to account for extraordinary values of J. Increases in J with stem height corresponded with reported decreases in E with height. The covariance between J and E suggests some regulation of bending curvature by adjustments in cross-sectional area. These results suggest that stems taper to maintain a uniform bending curvature and that when E is relatively constant within and among stems, diameter along the stem or across stems can be predicted from bending moment using a simple power function.     

An experiment-based algorithm for predicting the partitioning of unfolded peptides into phosphatidylcholine bilayer interfaces

Knowing the partitioning free energy of unfolded polypeptides into membrane interfaces is necessary for understanding membrane protein stability and for designing antimicrobial and other peptides. Experiment-based whole-residue free-energy (hydropathy) scales for amino acids in unfolded peptides, derived from the partitioning of host-guest pentapeptides (Ac-WLXLL) into the interfaces of phosphatidylcholine bilayers and into n-octanol, have been determined by W. C. Wimley, S. H. White, and colleagues [(1996) Nat. Struc. Biol. 3, 842; Wimley, W. C. et al. (1996) Biochemistry 35, 5109]. These scales offer the possibility of computing absolute partitioning free energies of unfolded peptides given only their amino acid sequences. However, the scales are incomplete, because partitioning free energies of N- and C-terminal groups are missing. To complete the scales, we have measured the pH-dependent partitioning of the host-guest pentapeptide variants AcWL-X-LL-NH(2) and WL-X-LL-NH(2) (X = G or W) into palmitoyloleoylphosphatidylcholine (POPC) bilayer interfaces and n-octanol. These measurements, in combination with the earlier ones, lead to hydrophobicity scale values for protonation, deprotonation, or acetylation of the N terminus and protonation, deprotonation, or amidation of the C terminus. A surprising finding is that a charged N terminus has a much smaller effect on bilayer partitioning than a charged C terminus. We present a simple algorithm for computing the absolute partitioning free energies of unfolded peptides into the phosphatidylcholine bilayer interface.     

Electronic Properties of the Amino Acid Side Chains Contribute to the Structural Preferences in Protein Folding

Abstract

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: α-helix, β-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (eσ), and a composite of these two scales (ε). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for α-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

A new amphipathy scale: I. Determination of the scale from molecular dynamics data

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl₄, were used resulting in the two AC_water and AC_CCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The AC_CCl4 scale is related with the size of side chain amino acids while AC_water is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where AC_water was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro.

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales

Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host-guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK-amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC-derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap-joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics-canada.org/TM/.     

Why is the biological hydrophobicity scale more accurate than earlier experimental hydrophobicity scales?

The recognition of transmembrane helices by the translocon is primarily guided by the average hydrophobicity of the potential transmembrane helix. However, the exact hydrophobicity of each amino acid can be identified in several different ways. The free energy of transfer for amino acid analogues between a hydrophobic media, for example, octanol and water can be measured or obtained from simulations, the hydrophobicity can also be estimated by statistical properties from known transmembrane segments and finally the contribution of each amino acid type for the probability of translocon recognition has recently been measured directly. Although these scales correlate quite well, there are clear differences between them and it is not well understood which scale represents neither the biology best nor what the differences are. Here, we try to provide some answers to this by studying the ability of different scales to recognize transmembrane helices and predict the topology of transmembrane proteins. From this analysis it is clear that the biological hydrophobicity scale as well scales created from statistical analysis of membrane helices perform better than earlier experimental scales that are mainly based on measurements of amino acid analogs and not directly on transmembrane helix recognition. Using these results we identified the properties of the scales that perform better than other scales. We find, for instance, that the better performing scales consider proline more hydrophilic. This shows that transmembrane recognition is not only governed by pure hydrophobicity but also by the helix preferences for amino acids, as proline is a strong helix breaker. Proteins 2014; 82:2190–2198. © 2014 Wiley Periodicals, Inc.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Electronic properties of the amino acid side chains contribute to the structural preferences in protein folding.

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: alpha-helix, beta-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (esigma), and a composite of these two scales (epsilon). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for alpha-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

APDbase: Amino acid Physico-chemical properties Database

Physico-chemical properties of amino acids can be used to study protein sequence profiles, folding and function. We collated 242 properties for the 20 naturally occurring amino acids and created a dataset. The dataset is available as a database named APDbase( Amino acid Physico-chemical properties Data base). The database can be queried using either key words describing physico-chemical properties or pre-assigned database index number. The database contains corresponding references for each property value and facilitates deposition of new property values for processing and inclusion in the database.

Availability     

Determinants of substrate affinity for the oligopeptide/H+ symporter in the renal brush border membrane.

We and others have shown previously the existence of high and low affinity systems for oligopeptide transport in kidney brush border membrane vesicles (BBMV). In the present study we investigated the relationship between the structure of substrates and their affinity for interaction with the high-affinity oligopeptide/H+ transporter in kidney BBMV. Based on competition experiments using [3H]Gly-Gln as a probe we determined the Ki values for more than 60 selected peptides. For a high-affinity interaction with the carrier site the following structural features of substrates are required: (a) both a free amino and carboxyl terminus; (b) the amino group and peptide bond nitrogen located in the alpha-position; (c) a trans peptide bond rather than the cis configuration; (d) L-alpha-amino acid isomers in both COOH and NH2 termini, although D-isomers of hydrophobic amino acids are acceptable in the NH2 terminus; and (e) a backbone of less than 3 amino acid residues. A striking finding of the present study is that, for peptides satisfying these minimal structural requirements, the primary determinant of affinity is hydrophobicity. The fact that there is a highly significant (p less than 0.001) correlation between Ki and hydrophobicity allows the prediction of the affinity for any di- or tripeptide composed of alpha-amino acids in the L-form.