2D IR Correlation Spectroscopy in the Determination of Aggregation and Stability of KH Domain GXXG Loop Peptide in the Presence and Absence of Trifluoroacetate

By Aslin Rodriguez Nassif, Igor de la Arada, Jose Luis R. Arrondo, Belinda Pastrana Rios

July 28, 2017

  • Year

  • Technique

  • Industry

Title

2D IR Correlation Spectroscopy in the Determination of Aggregation and Stability of KH Domain GXXG Loop Peptide in the Presence and Absence of Trifluoroacetate

Author

Aslin Rodriguez Nassif, Igor de la Arada, Jose Luis R. Arrondo, Belinda Pastrana Rios

Year

2017

Journal

Analytical Chemistry

Abstract

Trifluoroacetate (TFA) is a strong anion byproduct of solid phase peptide synthesis. Fourier transform infrared (FT-IR) spectroscopy can be used to ascertain the presence of this excipient in peptide samples for quality assessment. TFA absorbs as a strong sharp peak (1675.3 cm-1) within the amide I’ band of the spectral region. A peptide sample and the TFA excipient can be studied simultaneously by FT-IR and 2D IR correlation spectroscopies. In addition, these techniques are able to determine the effect of TFA on the stability of the peptide. Herein, we describe the spectroscopic characterization of the GXXG loop peptide (GXXGlp), which is present in KH domain containing proteins. The sequence of the Homo sapiens Krr1 GXXGlp is evolutionarily conserved (165KRRQRLIGPKGSTLKALELLTNCY189) and has been associated with ssDNA interaction and ribosome biogenesis. Our goal was to determine the structural elements present in this peptide and evaluate whether TFA affects the stability of GXXGlp during thermal stress. We observed differences in the molecular behavior of the synthetic peptide in the presence and absence of TFA at various peptide concentrations. The mechanism of aggregation was established by FT-IR at high peptide concentrations, but more importantly, we were able to define the optimum formulation conditions under which to study this peptide. Finally, 2D IR correlation spectroscopy was used for the determination of the unfolding process, mechanism and extent of peptide aggregation, and the effect of TFA on the stability of the peptide. This spectroscopic method can be applied to the characterization of any synthetic peptide.

Full Article

http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b04800

Instrument

J-810

Keywords

Circular dichroism, Secondary structure, Chemical stability, Biochemistry