A Novel Method to Measure Self-association of Small Amphipathic Molecules

Abstract

Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the selfassociation 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 highperformance 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) 1 /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...

Studies on Mechanism of Action of Anticancer Peptides by Modulation of Hydrophobicity Within a Defined Structural Framework

“…S1C) were defined as the peptide association parameter (P A ) to quantify the self-association ability of peptides in solution ( Table 1). The details of how to determine peptide association parameter (P A ) were reported previously (9,12,13). As seen from the data in Table 1, peptide association parameter (P A ) is not correlated with peptide relative hydrophobicity during RP-HPLC (t R ), especially for the Ala-substituted peptide analogues.…”

“…1A. An 18-mer control peptide (peptide C; Ac-ELEKGGLEGEKGGKE-LEK-amide) was used for temperature profiling during RP-HPLC to monitor peptide self-association ability as described previously (9,12,13).…”

“…Runs were done on a Zorbax 300 SB-C 8 column (150 Â 4.6-mm ID, 5-mm particle size, 300-A pore size) from Agilent Technologies, using a linear AB gradient (1% acetonitrile/min) and a flow rate of 1 mL/min, in which eluent A was 0.1% aqueous TFA and eluent B was 0.1% TFA in acetonitrile. Temperature profiling analyses during reversed phase (RP)-HPLC were done in 5 C increments, from 5 C to 80 C, as described previously (12,13).…”

Isothermal Titration Calorimetry Studies of the Binding of a Rationally Designed Analogue of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes

“…Note that the entropy term in eq 6 is replaced with a partial differential in surface pressure which follows from a twodimensional analogue of one of Maxwell relationships. The equation becomes (8) Note that the volume change in the condensed phase is negligible, i.e., ΔH = ΔE + Δ(PV) ≈ ΔE. This relation implies that the increase in surface area (ΔA) upon binding of the peptide to the bilayer surface amounts to an increase in ΔH.…”

Structure-Activity Relationships of Diastereomeric Lysine Ring Size Analogs of the Antimicrobial Peptide Gramicidin S

“…Retention times were recorded at temperatures between 5 and 80°C (3°C increments) using the temperature profiling RP-HPLC protocols described previously (25). 31 P NMR spectra were recorded with a Bruker MSL-400 solid state spectrometer operating at 161 MHz for 31 P, using data acquisition and data processing protocols similar to those described previously (12).…”