Two-Dimensional Infrared Correlation Analysis of Protein Unfolding: Use of Spectral Simulations to Validate Structural Changes during Thermal Denaturation of Bacterial CMP Kinases

Authors: Schultz, Christian P.1; Bârzu, Octavian2; Mantsch, Henry H.1

Source: Applied Spectroscopy, Volume 54, Issue 7, Pages 230A-260A and 931-1090 (July 2000) , pp. 931-938(8)

Publisher: Society for Applied Spectroscopy

Buy & download fulltext article:

OR

Price: $29.00 plus tax (Refund Policy)

Abstract:

The functional role of bacterial CMP kinases is to recover the energetically exhausted nucleoside monophosphates derived from cell metabolism by transferring a phosphate residue from ATP to CMP or dCMP. These enzymes-important for cell growth and division-possess two distinct binding sites and a number of conserved secondary structure elements. Herein we compare the infrared spectra of two similar, but not identical, CMP kinases from Escherichia coli and Bacillus subtilis. The two-dimensional correlation analysis of the infrared spectra of the two enzymes reveals significant differences in protein structure upon denaturation, a fact possibly linked to their different biochemical and catalytic properties. Model calculations are used to illustrate the effect of two separate processes on the out-of-phase correlation in the two-dimensional (2D) correlation plots. This strategy is then employed to validate the changes observed in the secondary structure of the two enzymes. When bound to the active site of the protein, the two substrates CMP and ATP exert a stabilizing effect on the structure of both proteins; however, the changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations that simulate the denaturation of the two enzymes confirm the occurrence of temperature-delayed unfolding processes in both proteins. Thermal denaturation and aggregation can be distinguished in both proteins as two distinct processes, separated in time.

Keywords: 2D-IR; FT-IR SPECTROSCOPY; PROTEIN FOLDING; PROTEIN SECONDARY STRUCTURE ANALYSIS; TWO-DIMENSIONAL IR CORRELATION SPECTROSCOPY

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702001950562

Affiliations: 1: Institute for Biodiagnostics, National Research Council of Winnipeg, Manitoba R3B 1Y6, Canada 2: Laboratoire de Chimie Structurale des Macromolecules, Institut Pasteur, 75724 Paris, Cedex 15, France

Publication date: July 1, 2000

More about this publication?
Related content

Tools

Key

Free Content
Free content
New Content
New content
Open Access Content
Open access content
Subscribed Content
Subscribed content
Free Trial Content
Free trial content

Text size:

A | A | A | A
Share this item with others: These icons link to social bookmarking sites where readers can share and discover new web pages. print icon Print this page