A novel method to measure self-association of small amphipathic molecules - Temperature profiling in reversed-phase chromatography

被引:61
作者
Lee, DL
Mant, CT
Hodges, RS
机构
[1] Univ Colorado, Hlth Sci Ctr, Dept Biochem & Mol Genet, Denver, CO 80262 USA
[2] Univ Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
关键词
D O I
10.1074/jbc.M301777200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 5-80degreesC 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.
引用
收藏
页码:22918 / 22927
页数:10
相关论文
共 51 条
[1]   KINETICS OF RECOMBINANT HUMAN BRAIN-DERIVED NEUROTROPIC FACTOR UNFOLDING UNDER REVERSED-PHASE LIQUID-CHROMATOGRAPHY CONDITIONS [J].
BENEDEK, K .
JOURNAL OF CHROMATOGRAPHY, 1993, 646 (01) :91-98
[2]   INDUCED CONFORMATIONAL STATES OF AMPHIPATHIC PEPTIDES IN AQUEOUS LIPID ENVIRONMENTS [J].
BLONDELLE, SE ;
OSTRESH, JM ;
HOUGHTEN, RA ;
PEREZPAYA, E .
BIOPHYSICAL JOURNAL, 1995, 68 (01) :351-359
[3]   Kinetic study on the formation of a de novo designed heterodimeric coiled-coil: Use of surface plasmon resonance to monitor the association and dissociation of polypeptide chains [J].
Chao, HM ;
Houston, ME ;
Grothe, S ;
Kay, CM ;
OConnorMcCourt, M ;
Irvin, RT ;
Hodges, RS .
BIOCHEMISTRY, 1996, 35 (37) :12175-12185
[4]   Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic α-helix [J].
Chen, Y ;
Mant, CT ;
Hodges, RS .
JOURNAL OF PEPTIDE RESEARCH, 2002, 59 (01) :18-33
[5]   Real-time monitoring of the interactions of two-stranded de novo designed coiled-coils:: Effect of chain length on the kinetic and thermodynamic constants of binding [J].
De Crescenzo, G ;
Litowski, JR ;
Hodges, RS ;
O'Connor-McCourt, MD .
BIOCHEMISTRY, 2003, 42 (06) :1754-1763
[6]   Unusual β-sheet periodicity in small cyclic peptides [J].
Gibbs, AC ;
Kondejewski, LH ;
Gronwald, W ;
Nip, AM ;
Hodges, RS ;
Sykes, BD ;
Wishart, DS .
NATURE STRUCTURAL BIOLOGY, 1998, 5 (04) :284-288
[7]   REVERSED-PHASE LIQUID-CHROMATOGRAPHY AS A USEFUL PROBE OF HYDROPHOBIC INTERACTIONS INVOLVED IN PROTEIN-FOLDING AND PROTEIN STABILITY [J].
HODGES, RS ;
ZHU, BY ;
ZHOU, NE ;
MANT, CT .
JOURNAL OF CHROMATOGRAPHY A, 1994, 676 (01) :3-15
[8]   DENATURATION AND THE EFFECTS OF TEMPERATURE ON HYDROPHOBIC-INTERACTION AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF PROTEINS - BIO-GEL TSK-PHENYL-5-PW COLUMN [J].
INGRAHAM, RH ;
LAU, SYM ;
TANEJA, AK ;
HODGES, RS .
JOURNAL OF CHROMATOGRAPHY, 1985, 327 (JUN) :77-92
[9]   Diastereoisomeric analogues of gramicidin S: structure, biological activity and interaction with lipid bilayers [J].
Jelokhani-Niaraki, M ;
Kondejewski, LH ;
Farmer, SW ;
Hancock, REW ;
Kay, CM ;
Hodges, RS .
BIOCHEMICAL JOURNAL, 2000, 349 :747-755
[10]   Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers [J].
Jelokhani-Niaraki, M ;
Prenner, EJ ;
Kay, CM ;
McElhaney, RN ;
Hodges, RS .
JOURNAL OF PEPTIDE RESEARCH, 2002, 60 (01) :23-36