A Novel Method to Measure Self-association of Small Amphipathic Molecules
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m301777200
ISSN1083-351X
AutoresDarin L. Lee, Colin T. Mant, Robert S. Hodges,
Tópico(s)Protein purification and stability
ResumoBiophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the self-association of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5–80 °C during gradient elution reversed-phase high-performance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution. Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the self-association of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5–80 °C during gradient elution reversed-phase high-performance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution. The detection of molecular self-association and aggregation is an important concern for molecules intended for biological applications, such as proteins, peptides, and small organic drug molecules. A plethora of biophysical techniques already exist for the detection of molecular self-association in aqueous solution, including spectroscopic (NMR, CD, Fourier-transform infrared spectroscopy (FTIR), fluorescence, light scattering), chromatographic (affinity, size-exclusion (SEC) 1The abbreviations used are: SEC, size exclusion chromatography; RP-HPLC, reversed-phase high performance liquid chromatography; lin, linear. /gel filtration), and other techniques (matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), non-denaturing PAGE, sedimentation equilibrium analytical ultracentrifugation). However, some of the classical techniques commonly used for measuring self-association (sedimentation equilibrium analytical ultracentrifugation, non-denaturing PAGE, and size-exclusion chromatography) possess a lower limit to the molecular size that can be clearly resolved, such that fewer methods exist for detecting self-association in smaller molecules. With a view to solving this problem, we describe here a novel method for measuring self-association, referred to as "temperature profiling in reversed-phase chromatography," based on observation of the conformation-dependent response of peptides to RP-HPLC under changing temperature. Much of the efficacy of RP-HPLC as a probe of polypeptide stability, folding, and conformation lies in the extensive range of stationary phases and/or mobile phase conditions available to the researcher when relating polypeptide elution behavior with structural features (e.g. the amphipathicity of α-helices or cyclic β-sheet peptides; destabilization of conformation) (1Mant C.T. Zhou N.E. Hodges R.S. J. Chromatogr. 1989; 476: 363-375Crossref PubMed Scopus (96) Google Scholar, 2Rosenfeld R. Benedek K. J. Chromatogr. 1993; 632: 29-36Crossref PubMed Scopus (32) Google Scholar, 3Benedek K. J. Chromatogr. 1993; 646: 91-98Crossref PubMed Scopus (18) Google Scholar, 4Hodges R.S. Zhu B.-Y. Zhou N.E. Mant C.T. J. Chromatogr. A. 1994; 676: 3-15Crossref PubMed Scopus (53) Google Scholar, 5Richards K.L. Aguilar M.I. Hearn M.T.W. J. Chromatogr. 1994; 676: 17-31Crossref Scopus (25) Google Scholar, 6Yu Y.B. Wagschal K.C. Mant C.T. Hodges R.S. J. Chromatogr. A. 2000; 890: 81-94Crossref PubMed Scopus (14) Google Scholar, 7Mant C.T. Litowski J.R. Hodges R.S. J. Chromatogr. A. 1998; 816: 65-78Crossref PubMed Scopus (64) Google Scholar, 8Mant C.T. Kondejewski L.H. Hodges R.S. J. Chromatogr. A. 1998; 816: 79-88Crossref Scopus (50) Google Scholar, 9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 10Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Crossref PubMed Scopus (211) Google Scholar, 11Chen Y. Mant C.T. Hodges R.S. J. Peptide Res. 2002; 59: 18-33Crossref PubMed Scopus (84) Google Scholar) and/or biological activity (e.g. antimicrobial potency, receptor binding) (12Sereda T.J. Mant C.T. Sonnichsen F.D. Hodges R.S. J. Chromatogr. A. 1994; 676: 139-153Crossref PubMed Scopus (86) Google Scholar, 13Mant C.T. Hodges R.S. J. Chromatogr. A. 2002; 972: 45-60Crossref PubMed Scopus (14) Google Scholar, 14Mant C.T. Hodges R.S. J. Chromatogr. A. 2002; 972: 61-75Crossref PubMed Scopus (12) Google Scholar). Such studies are based on the premise that the hydrophobic interactions between polypeptides and the nonpolar stationary phase characteristic of RP-HPLC (15Mant C.T. Hodges R.S. Mant C.T. Hodges R.S. High-Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation. CRC Press, Boca Raton, FL1991Google Scholar, 16Mant C.T. Hodges R.S. Gooding K.M. Regnier F.E. HPLC of Biological Macromolecules: Methods and Applications. 2nd Ed. Marcel Dekker, Inc., New York2002: 433-511Google Scholar, 17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) mimic the hydrophobicity and interactions between nonpolar residues, which are the major driving forces for protein folding and stability, including those dictating the level and stability of polypeptide oligomerization. We believe temperature adds another dimension to such applications, with physicochemical studies of RP-HPLC of polypeptide solutes under conditions of varying temperature (hence, "temperature profiling") allowing even more insight into conformational stability and self-association of peptide solutes. The present study demonstrates the utility of temperature profiling in RP-HPLC to monitor self-association of small cyclic peptides based on the de novo-designed amphipathic cytolytic peptide, GS14, through the interpretation of peptide elution behavior over a temperature range of 5–80 °C. This approach is shown to be simple to operate, highly sensitive, requires low sample quantities (nanograms to low micrograms) and is capable of analyzing small molecules (<2000 Da). In addition, the applicability of this novel approach for other small molecules is also discussed. Peptide Synthesis, Purification, and Cyclization—Linear and cyclic peptides were synthesized using standard t-butyloxycarbonyl chemistry, as described previously (17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). N-terminal and C-terminal groups of linear peptides were left as free amino and carboxyl groups, respectively. Peptides were cleaved using standard HF protocol and purified by reversed-phase chromatography. Peptides intended for cyclization were cyclized, deformylated, and purified using the method of Kondejewski et al. (17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Analytical Reversed-phase Analysis of Peptides—Peptides were analyzed on an Agilent 1100 Series liquid chromatograph (Little Falls, DE). Runs were performed on a Zorbax 300SB-C8 column (2.1 mm (inner diameter) × 150 mm, 300-Å pore size, 5-μm particle size) from Agilent Technologies using a linear AB gradient (0.5% B/min) and a flow rate of 0.35 ml/min, where solvent A is 0.05% aqueous trifluoroacetic acid, pH 2 and solvent B is 0.05% trifluoroacetic acid in acetonitrile. Analyses were performed in 5 °C increments, from 5 to 80 °C. CD Spectroscopy—CD measurements were obtained on a JASCO J-810 Spectropolarimeter at 20 °C (Easton, MD) using Spectra Manager 228 software, Version 1.10.00 running on a Pentium III under Microsoft Windows 2000 (Redmond, WA). Temperature was controlled in a Lauda Model RMS-6 water bath (Brinkman Instruments, Rexdale, Canada). Peptide concentrations used in this study were ∼30 μm as determined by amino acid analysis or UV absorbance at 280 nm (ϵ = 2840 cm–1m–1 from two d-Tyr residues) of stock solutions. Data were collected at 0.1-nm intervals from 190 to 250 nm for wavelength scans at 20 °C, with the average of 4 or 8 scans reported. Benign buffer (i.e. nondenaturing to peptide structure) was 5 mm sodium acetate, pH 5.5. Scans were also performed in 5 mm sodium acetate, pH 5.5 containing 50% (v/v) trifluoroethanol. Ellipticity was reported as mean residue molar ellipticity ([θ]) in deg·cm2·dmol–1 using Equation 1, [θ]=θ(MRW)/10lc (Eq. 1) where θ is the ellipticity in millidegrees, MRW is the mean residue weight (molecular weight divided by number of residues), l is the optical path length of the cell in centimeters, and c is the peptide concentration in mg/ml. Alternatively, for ease of use, the equation can be rearranged to Equation 2, [θ]=θ/10lcMn (Eq. 2) where θ is the ellipticity in millidegrees, l is the optical path length of the cell in centimeters, cM is the peptide concentration in mol/liter, and n is the number of residues in the peptide. This form allows direct entry of ellipticity (in millidegrees) and concentration (in molarity) values obtained experimentally, without requiring the mean residue weight calculation. Curve Fitting—Temperature profiling data were fit to either a first-order nonlinear least-squares equation, a fourth-order polynomial, or a smooth fit using Kaleidagraph v. 3.52 (Synergy Software, Reading, PA). Transition temperature values (Tp) were defined as the temperatures at which the maximum retention times were experimentally observed, i.e. in 5 °C-increments, and were not determined by maxima obtained from fitted curves. If more accurate data are required, we recommend that the analysis be performed in 3 °C increments from 5 to 80 °C if sample quantity and run time are not limiting factors. Parent Peptide (GS14)—GS14 is a 14-residue cytolytic amphipathic peptide designed de novo in our laboratory (Fig. 1). The sequence of GS14 was originally developed as an analog of the cationic antimicrobial peptide gramicidin S (17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), whose activity is thought to be a result of interaction with lipid membranes (9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 18Katsu T. Kuroko M. Morikawa T. Sanchika K. Fujita Y. Yamamura H. Uda M. Biochim. Biophys. Acta. 1989; 983: 135-141Crossref PubMed Scopus (130) Google Scholar, 19Prenner E.J. Lewis R.N. Neuman K.C. Gruner S.M. Kondejewski L.H. Hodges R.S. McElhaney R.N. Biochemistry. 1997; 36: 7906-7916Crossref PubMed Scopus (123) Google Scholar, 20Prenner E.J. Lewis R.N. McElhaney R.N. Biochim. Biophys. Acta. 1999; 1462: 201-221Crossref PubMed Scopus (200) Google Scholar, 21Prenner E.J. Lewis R.N. Kondejewski L.H. Hodges R.S. McElhaney R.N. Biochim Biophys Acta. 1999; 1417: 211-223Crossref PubMed Scopus (116) Google Scholar, 22Lewis R.N. Prenner E.J. Kondejewski L.H. Flach C.R. Mendelsohn R. Hodges R.S. McElhaney R.N. Biochemistry. 1999; 38: 15193-15203Crossref PubMed Scopus (54) Google Scholar, 23Jelokhani-Niaraki M. Kondejewski L.H. Farmer S.W. Hancock R.E. Kay C.M. Hodges R.S. Biochem. J. 2000; 349 Pt 3: 747-755Crossref PubMed Scopus (60) Google Scholar, 24Kondejewski L.H. Lee D.L. Jelokhani-Niaraki M. Farmer S.W. Hancock R.E. Hodges R.S. J. Biol. Chem. 2002; 277: 67-74Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 25Jelokhani-Niaraki M. Prenner E.J. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2002; 60: 23-36Crossref PubMed Scopus (37) Google Scholar). GS14 has been previously studied by a number of techniques, including RP-HPLC (9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 26Jelokhani-Niaraki M. Prenner E.J. Kondejewski L.H. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2001; 58: 293-306Crossref PubMed Scopus (32) Google Scholar), CD spectroscopy (9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 24Kondejewski L.H. Lee D.L. Jelokhani-Niaraki M. Farmer S.W. Hancock R.E. Hodges R.S. J. Biol. Chem. 2002; 277: 67-74Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 25Jelokhani-Niaraki M. Prenner E.J. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2002; 60: 23-36Crossref PubMed Scopus (37) Google Scholar, 26Jelokhani-Niaraki M. Prenner E.J. Kondejewski L.H. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2001; 58: 293-306Crossref PubMed Scopus (32) Google Scholar), NMR spectroscopy (9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 27McInnes C. Kondejewski L.H. Hodges R.S. Sykes B.D. J. Biol. Chem. 2000; 275: 14287-14294Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), UV absorption, and fluorescence spectroscopy (26Jelokhani-Niaraki M. Prenner E.J. Kondejewski L.H. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2001; 58: 293-306Crossref PubMed Scopus (32) Google Scholar). It is a cyclic β-sheet molecule that contains six aliphatic residues (3 Val and 3 Leu) on the nonpolar face and four basic (Lys) residues on the polar face; the segregation of nonpolar and charged residues on opposite sides of the molecule makes it an extremely amphipathic peptide (Fig. 2A) (27McInnes C. Kondejewski L.H. Hodges R.S. Sykes B.D. J. Biol. Chem. 2000; 275: 14287-14294Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). GS14 is also a suitable choice for de novo peptide design and structure-activity studies because the cyclic nature of the peptide prevents amino acid substitutions from significantly altering secondary structure, i.e. switching the β-sheet to an α-helix or random coil. Previous studies indicated that GS14 aggregated above 50 μm peptide concentration in aqueous solution (26Jelokhani-Niaraki M. Prenner E.J. Kondejewski L.H. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2001; 58: 293-306Crossref PubMed Scopus (32) Google Scholar). Unfortunately, attempts to determine the oligomerization state failed by sedimentation equilibrium analytical ultracentrifugation (because of the low molecular weight) and by size-exclusion chromatography (because of high sample load requirements and low peptide solubility), thus emphasizing the need for an alternative technique to examine self-association of this molecule. GS14 is biologically active against human red blood cells (hemolytic); however, it shows little or no antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and yeast (17Kondejewski L.H. Farmer S.W. Wishart D.S. Kay C.M. Hancock R.E. Hodges R.S. J. Biol. Chem. 1996; 271: 25261-25268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).Fig. 2NMR structures and Connolly surface representations of GS14 and GS14K4 peptides (27McInnes C. Kondejewski L.H. Hodges R.S. Sykes B.D. J. Biol. Chem. 2000; 275: 14287-14294Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Connolly surface representations are rotated clock-wise 90° along the X-axis relative to the NMR structures. Amino acid side chains are labeled with one-letter code and sequence number, with Lys-4 stereochemistry designated in subscript. Oxygen atoms are red; nitrogen atoms are blue. Panel A, structure of GS14 (left) showing the positioning of the nonpolar residues (3 Leu and 3 Val) on one face and the four Lys residues on the other face and the Connolly surface representation (right) showing the nonpolar face. Panel B, structure of GS14K4 (left) and the Connolly surface representation (right) showing the position of the d-Lys at position 4 on the nonpolar face.View Large Image Figure ViewerDownload (PPT) RP-HPLC of Amphipathic Peptides—It is well documented that the formation of a hydrophobic binding domain due to peptide secondary structure can affect peptide interactions with reversed-phase matrices, this effect having been observed both for amphipathic α-helical peptides (4Hodges R.S. Zhu B.-Y. Zhou N.E. Mant C.T. J. Chromatogr. A. 1994; 676: 3-15Crossref PubMed Scopus (53) Google Scholar, 6Yu Y.B. Wagschal K.C. Mant C.T. Hodges R.S. J. Chromatogr. A. 2000; 890: 81-94Crossref PubMed Scopus (14) Google Scholar, 7Mant C.T. Litowski J.R. Hodges R.S. J. Chromatogr. A. 1998; 816: 65-78Crossref PubMed Scopus (64) Google Scholar, 11Chen Y. Mant C.T. Hodges R.S. J. Peptide Res. 2002; 59: 18-33Crossref PubMed Scopus (84) Google Scholar, 12Sereda T.J. Mant C.T. Sonnichsen F.D. Hodges R.S. J. Chromatogr. A. 1994; 676: 139-153Crossref PubMed Scopus (86) Google Scholar, 13Mant C.T. Hodges R.S. J. Chromatogr. A. 2002; 972: 45-60Crossref PubMed Scopus (14) Google Scholar, 14Mant C.T. Hodges R.S. J. Chromatogr. A. 2002; 972: 61-75Crossref PubMed Scopus (12) Google Scholar, 28Zhou N.E. Mant C.T. Hodges R.S. Pept. Res. 1990; 3: 8-20PubMed Google Scholar, 29Mant C.T. Zhou N.E. Hodges R.S. Epand R.M. The Amphipathic Helix. CRC Press, Inc., Boca Raton, FL1993: 39-64Google Scholar) and amphipathic, cyclic β-sheet peptides (8Mant C.T. Kondejewski L.H. Hodges R.S. J. Chromatogr. A. 1998; 816: 79-88Crossref Scopus (50) Google Scholar, 9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Thus, peptides containing such preferred binding domains will exhibit significantly greater retention times compared with analogs with the same amino acid composition (i.e. the same intrinsic hydrophobicity) but lacking such a domain (8Mant C.T. Kondejewski L.H. Hodges R.S. J. Chromatogr. A. 1998; 816: 79-88Crossref Scopus (50) Google Scholar, 28Zhou N.E. Mant C.T. Hodges R.S. Pept. Res. 1990; 3: 8-20PubMed Google Scholar). Indeed, the chromatography conditions characteristic of RP-HPLC (hydrophobic stationary phase, nonpolar eluting solvent) are able to induce and stabilize secondary structure in both potentially α-helical (28Zhou N.E. Mant C.T. Hodges R.S. Pept. Res. 1990; 3: 8-20PubMed Google Scholar, 30Steiner V. Scharr M. Bornsen K.O. Mutter M. J. Chromatogr. 1991; 586: 43-50Crossref PubMed Scopus (48) Google Scholar, 31Purcell A.W. Aguilar M.I. Wettenhall R.E.W. Hearn M.T.W. Peptide Res. 1995; 8: 160-170PubMed Google Scholar, 32Blondelle S.E. Ostresh J.M. Houghten R.A. Perez-Paya E. Biophys. J. 1995; 68: 351-359Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 33Steer D.L. Thompson P.E. Blondelle S.E. Houghten R.A. Aguilar M.I. J. Peptide Res. 1998; 51: 401-412Crossref PubMed Scopus (32) Google Scholar) and cyclic, β-sheet (33Steer D.L. Thompson P.E. Blondelle S.E. Houghten R.A. Aguilar M.I. J. Peptide Res. 1998; 51: 401-412Crossref PubMed Scopus (32) Google Scholar) peptides; concomitantly, tertiary and quaternary structure is disrupted by such conditions (1Mant C.T. Zhou N.E. Hodges R.S. J. Chromatogr. 1989; 476: 363-375Crossref PubMed Scopus (96) Google Scholar, 12Sereda T.J. Mant C.T. Sonnichsen F.D. Hodges R.S. J. Chromatogr. A. 1994; 676: 139-153Crossref PubMed Scopus (86) Google Scholar, 28Zhou N.E. Mant C.T. Hodges R.S. Pept. Res. 1990; 3: 8-20PubMed Google Scholar, 34Lau S.Y. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 259: 13253-13261Abstract Full Text PDF PubMed Google Scholar, 35Lau S.Y.M. Taneja A.K. Hodges R.S. J. Chromatogr. 1984; 317: 129-140Crossref Scopus (154) Google Scholar, 36Ingraham R.H. Lau S.Y.M. Taneja A.K. Hodges R.S. J. Chromatogr. 1985; 327: 77-92Crossref Scopus (111) Google Scholar, 37Mant C.T. Chao H. Hodges R.S. J. Chromatogr. A. 1997; 791: 85-98Crossref PubMed Scopus (19) Google Scholar). Due to the intimate interaction of the nonpolar faces of amphipathic cyclic analogs with a reversed-phase matrix, any differences in effective hydrophobicity of these preferred binding domains via amino acid substitutions in this domain will be readily monitored through subsequent differences in RP-HPLC retention time behavior. Peptide Diastereomer (GS14K4)—Because GS14 has such low specificity toward microbial membranes, analogs of GS14 have been studied in an attempt to reverse this activity profile and to create an antimicrobial peptide with minimal toxicity to human cells and high activity toward pathogens. One such analog is GS14K4 (Fig. 2B), a diastereomer of GS14 with the d-enantiomer of l-Lys substituted at position 4 (9Kondejewski L.H. Jelokhani-Niaraki M. Farmer S.W. Lix B. Kay C.M. Sykes B.D. Hancock R.E. Hodges R.S. J. Biol. Chem. 1999; 274: 13181-13192Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Kondejewski L.H. Lee D.L. Jelokhani-Niaraki M. Farmer S.W. Hancock R.E. Hodges R.S. J. Biol. Chem. 2002; 277: 67-74Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). While the positively charged l-Lys 4 in GS14 is normally on the polar face, the d-Lys 4 in GS14K4 is positioned on the nonpolar face and disrupts the β-sheet structure in benign medium. GS14K4 exhibits an activity profile opposite to that of GS14 with a 103- to 104-fold improvement in therapeutic index (a measure of peptide specificity for microbial cells over human cells). Since the highly hydrophilic d-Lys is positioned on the nonpolar face of the peptide, the amphipathicity of the molecule is significantly decreased, thus decreasing its retention time in RP-HPLC relative to GS14 by over 23 min at 80 °C (GS14, 85.6 min; GS14K4, 62.0 min, Table I). A comparison of the effect of temperature on the elution behavior of GS14 and GS14K4 is shown in Fig. 3. While GS14 aggregates at concentrations above 50 μm (84 μg/ml) in solution due to the high hydrophobicity of its nonpolar face, no aggregation was observed in GS14K4 up to the highest concentration studied, 175 μm (292 μg/ml) (26Jelokhani-Niaraki M. Prenner E.J. Kondejewski L.H. Kay C.M. McElhaney R.N. Hodges R.S. J. Pept. Res. 2001; 58: 293-306Crossref PubMed Scopus (32) Google Scholar). Because GS14 and GS14K4 possess the ability to be analyzed in an RP-HPLC system and exhibit marked differences in activity, structure, and aggregation phenomena, these de novo-designed cyclic peptides are ideal candidates for the investigation of relationships between molecular self-association and biological activity using the RP-HPLC technique of temperature profiling.Table IHPLC data for peptides used in this studyPeptidetR(80 °C)aPeptide retention time at 80 °C as determined by reversed-phase HPLC (see "Materials and Methods").tR(5 °C)bPeptide retention time at 5 °C as determined by reversed-phase HPLC (see "Materials and Methods").TPcTemperature at which the maximum retention time is observed over the temperature range 5-80 °C.tR(TP)dPeptide retention time at temperature TP.tR(TP)-tR(5 °C)eDifference in retention time between tR at the TP and tR at 5 °C.tR(TP)-tR(80 °C)fDifference in retention time between tR at the TP and tR at 80 °C.minmin°CminminminGS1485.682.55586.74.21.1GS14XD4 analogsGS14 LD4100.496.855101.85.01.4GS14 FD498.394.855100.05.21.7GS14 AD494.190.65595.34.71.2GS14 YD483.083.24085.11.92.1GS14 ND473.373.54075.31.82.0GS14 KD4gGS14KD4 and GS14K4 denote the same peptide with D-Lys at position 4.62.065.02065.30.33.3GS14K4 hydrophobicity analogsGS14K462.065.02065.30.33.3GS14K4 V3/A347.452.5552.505.1GS14K4 L3/A339.846.4546.406.6GS14K4 A625.833.3533.307.5Linear analogsGS14 lin55.662.81563.10.37.2GS14K4 lin46.251.6551.605.4GS14K4 A6 lin21.330.7530.709.4a Peptide retention time at 80 °C as determined by reversed-phase HPLC (see "Materials and Methods").b Peptide retention time at 5 °C as determined by reversed-phase HPLC (see "Materials and Methods").c Temperature at which the maximum retention time is observed over the temperature range 5-80 °C.d Peptide retention time at temperature TP.e Difference in retention time between tR at the TP and tR at 5 °C.f Difference in retention time between tR at the TP and tR at 80 °C.g GS14KD4 and GS14K4 denote the same peptide with D-Lys at position 4. Open table in a new tab GS14 XD4 Analogs—The GS14 XD4 analogs replaced the d-Lys in GS14K4 with a series of other d-amino acids at position 4 (Fig. 1). The substituted amino acids ranged in hydrophobicity from d-Asn and d-Tyr (least hydrophobic) to d-Leu and d-Phe (most hydrophobic) (12Sereda T.J. Mant C.T. Sonnichsen F.D. Hodges R.S. J. Chromatogr. A. 1994; 676: 139-153Crossref PubMed Scopus (86) Google Scholar, 38Monera O.D. Sereda T.J. Zhou N.E. Kay C.M. Hodges R.S. J. Pept. Sci. 1995; 1: 319-329Crossref PubMed Scopus (289) Google Scholar) and altered both the overall hydrophobicity and amphipathicity of the peptide caused by the substituted amino acid being positioned on the nonpolar face. The analogs also had a lower net charge versus GS14K4 (+3 instead of +4 from the Lys residues in GS14, Fig. 2). The differences in the peptide composition influenced peptide retention time in RP-HPLC, with the most hydrophobic amino acid substitution (d-Leu in GS14LD4) producing the largest retention time, compared with GS14K4, which had the least hydrophobic amino acid (d-Lys) at position 4 and the lowest retention time within the series. Retention times at 80 °C ranged from 62 to 100 min for d-Lys and d-Leu peptides, respectively (Table I). GS14K4 Hydrophobicity Analogs—To investigate the role of peptide hydrophobicity in microbial specificity, analogs of GS14K4 were previously created with variations of the aliphatic residues in the nonpolar face (24Kondejewski L.H. Lee D.L. Jelokhani-Niaraki M. Farmer S.W. Hancock R.E. Hodges R.S. J. Biol. Chem. 2002; 277: 67-74Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A subset of these hydrophobicity analogs was now assessed for self-association in the present study (Fig. 1). The selected analogs had lower nonpolar face hydrophobicity relative to GS14K4, with valines and/or leucines in GS14K4 substituted with the less hydrophobic alanine residue (12Sereda T.J. Mant C.T. Sonnichsen F.D. Hodges R.S. J. Chromatogr. A. 1994; 676: 139-153Crossref PubMed Scopus (86) Google Scholar, 38Monera O.D. Sereda T.J. Zhou N.E. Kay C.M. Hodges R.S. J. Pept. Sci. 1995; 1: 319-329Crossref PubMed Scopus (289) Google Scholar), e.g. peptide GS14K4 V3/A3
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