Thermal and chemical denaturation of Bacillus circulans xylanase

Abstract

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate DH o , DS o , and DG o T for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of DG o on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab.Keywords: Protein thermal and chemical denaturation, van't Hoff analysis, biophysical methods, laboratory exercises, protein structure, function, folding.The University of Richmond is a private liberal arts college of 2,800 undergraduates, which offers both B.A. and B.S. degrees in biochemistry and molecular biology, an interdisciplinary program that brings together faculty from both biology and chemistry. Begun officially in 2003, this program continues to grow, and last year a high of 28 majors graduated. As part of the major, students take a two-semester sequence with lab in biochemistry, which is usually done in either their junior or senior years. The second semester course is Proteins: Structure, Function, and Biophysics, which has 150 min of lecture (2 3 75 min classes) and 180 min of lab (one meeting, capped at 16 students) per week. Multiple faculties rotate through this course, each providing a slightly different focus with different labs. In one version of this course, the focus is on protein folding, modeled after that originally described by Anthony-Cahill [1]. What is described herein are two labs, one on protein chemical denaturation and one on protein thermal denaturation, which are valuable in reinforcing lecture concepts. These labs fit well into a previously described module focused on protein overexpression, purification, and secondary structure determination [2] (see instructor's notes).The lecture portion of the proteins course is divided into three parts: protein spectroscopy, protein thermodynamics, and protein primary literatur...

Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approach

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Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

“…This parameter can be determined by observing a protein's structural signal, such as circular dichroism, fluorescence, or NMR signal, as a function of temperature. Experimental determination of T m can be performed within a time frame of a typical undergraduate lab ( Raabe and Gentile, 2008 ). T m is simply the half point of the transition for a protein that folds in a cooperative two-state manner ( Figure 4 ).…”