Chapter 22
Cold Denaturation of Proteins Jiri Jonas
Downloaded by UNIV OF GUELPH LIBRARY on October 4, 2012 | http://pubs.acs.org Publication Date: September 30, 1997 | doi: 10.1021/bk-1997-0676.ch022
Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, IL 61801
By taking advantage of the phase behavior of water, high pressure can significantly lower the freezing point of an aqueous protein solution. In this way, using high-resolution, high-pressure N M R techniques, one can investigate not only pressure denaturation but also cold denaturation of proteins. After an overview of compression effects on dynamic and hydrodynamic behavior of water and heavy water at subzero temperatures, the main part of this contribution is devoted to selected results from recent pressure and cold denaturation N M R studies of Ribonuclease A . The cold denatured state of Ribonuclease A contains partial secondary structures in contrast to its thermally denatured state which contains little or no stable hydrogen bond structures. It was interesting to find that the pattern of protection factors for the pressure and cold denatured states of Ribonuclease A obtained by hydrogen exchange experiments parallels the pattern of protection factors for the folding intermediate of Ribonuclease A reported by Udgoaonkar and Baldwin on the basis of their pulsed hydrogen experiments.
Increasing attention has recently been focused on denatured and partially folded states, since determination of their structure and stability .may provide novel information for the mechanisms of protein folding (1-3). The native conformations of hundreds of proteins are known in great detail from structural determinations by X-ray crystallography and, more recently, N M R spectroscopy. However, a detailed knowledge of the conformations of denatured and partially folded states is lacking, and represents a serious shortcoming in current studies of protein stability and protein folding pathways (2). Protein folding, the relationship between the amino acid sequence and the 310
© 1997 American Chemical Society
In Supercooled Liquids; Fourkas, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by UNIV OF GUELPH LIBRARY on October 4, 2012 | http://pubs.acs.org Publication Date: September 30, 1997 | doi: 10.1021/bk-1997-0676.ch022
22. JONAS
Cold Denaturation ofProteins
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structure and dynamic properties of the native conformation of proteins, represents the central problem of biochemistry and biotechnology. Most studies dealing with protein denaturation have been carried out at atmospheric pressure using various physicochemical perturbations, such as temperature, pH, or denaturants, as experimental variables. Compared to varying temperature, which produces simultaneous changes in both volume and thermal energy, the use of pressure to study protein solutions perturbs the environment of the protein in a continuous, controlled way by changing only intermolecular distances. In addition, by taking advantage of the phase behavior of water high pressure can substantially lower the freezing point of an aqueous protein solution. Therefore, by applying high pressure one can investigate in detail not only pressure-denatured proteins, but also colddenatured proteins (4) in aqueous solution. Cold denaturation has been assumed to be a general property of all globular proteins (4, 5). However, experimental evidence for cold denaturation has been scant, owing to the fact that cold denaturation of proteins in aqueous solution is usually only observed at temperatures below 0°C at neutral pH. Different approaches have been utilized to prevent freezing of protein solutions, including the use of cryo-solvents (
