Temperature dependent 2nd derivative absorbance spectroscopy of aromatic amino acids as a probe of protein dynamics.
Proteins display a broad peak in 250-300 nm region of their UV spectrum containing multiple overlapping bands arising from the aromatic rings of phenylalanine, tyrosine, and tryptophan residues. Employing high resolution 2(nd) derivative absorbance spectroscopy, these overlapping absorption bands can be highly resolved and therefore provide a very sensitive measure of changes in the local microenvironment of the aromatic side chains. This has traditionally been used to detect both subtle and dramatic (i.e., unfolding) conformational alterations of proteins. Herein, we show that plots of the temperature dependent 2(nd) derivative peak positions of aromatic residues have measurable slopes before protein unfolding and that these slopes are sensitive to the dielectric properties of the surrounding microenvironment. We further demonstrate that these slopes correlate with hydration of the buried aromatic residues in protein cores and can therefore be used as qualitative probes of protein dynamics.
Esfandiary
Reza
R
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
Hunjan
Jagtar S
JS
Lushington
Gerald H
GH
Joshi
Sangeeta B
SB
Middaugh
C Russell
CR
D024322:Amino Acids, Aromatic / Q000032:analysis*; D017510:Protein Folding; D011506:Proteins / Q000032:analysis*; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013696:Temperature
D024322:Amino Acids, Aromatic; D011506:Proteins; D012997:Solvents