Temperature dependent 2^nd derivative absorbance spectroscopy of aromatic amino acids as a probe of protein dynamics

Abstract

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.

Linking the Solution Viscosity of an IgG2 Monoclonal Antibody to Its Structure as a Function of pH and Temperature

“…c) drifted slightly at lower temperatures and then showed distinct transitions. The linear shift in peak positions was also seen in model phenylalanine, tyrosine, and tryptophan compounds, and the slope of these peaks has been shown to reflect changes in the dynamic properties of proteins . At pH 4.0, there were two notable structural transitions as a function of temperature, the first transition starting at approximately 30°C, followed by a second that initiates at approximately 50°C and ends at 75°C.…”

“…Partial specific volume has been found to have a reasonably good correlation with the compressibility, that is, fluctuations and relaxations of a protein molecule in solution . Although we have found no previous attempt to use partial specific volume values to directly probe protein dynamic properties, the linear change of partial specific volume does correlate well with the temperature and the initial shift of peak positions of aromatic residues in the second‐derivative UV absorption spectra, in which the slope of the shift of the peak position was considered to be a measure of dynamic aspects of protein behavior …”

Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

“…13 Some protein has been crystallized through collaboration with a local research center using a mosquito crystallization robot (TTP Labtech). In semester courses these analyses could be integrated into the course or students could design proteins for investigation, producing expression plasmids through gene synthesis.…”

Subunit Vaccine Delivery

“…Some additional information can sometimes be obtained by perturbing proteins with variable conditions of pH and temperature. Information about protein dynamic structure can also be obtained from the pre-transition slope of a second derivate peak (in a wavelength versus temperature plot) (Esfandiary et al 2009 ) and shifts in peak position induced by added cations (Lucas et al 2006 ). In nucleic acids, the spectra of the bases are very sensitive to their local intramolecular interactions and this can be used for detection of secondary structure changes.…”