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Protein thermal stability, hydrogen bonds, and ion pairs1

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Abstract

Researchers in both academia and industry have expressed strong interest in comprehending the mechanisms responsible for enhancing the thermostability of proteins. Many and different structural principles have been postulated for the increased stability. Here, 16 families of proteins with different thermal stability were theoretically examined by comparing their respective fractional polar atom surface areas and the number and type of hydrogen bonds and salt links between explicit protein atoms. In over 80% of the families, correlations were found between the thermostability of the familial members and an increase in the number of hydrogen bonds as well as an increase in the fractional polar surface which results in added hydrogen bonding density to water. Thus increased hydrogen bonding may provide the most general explanation for thermal stability in proteins. The number of ion pairs was also found to increase with thermal stability in two-thirds of the families tested; however, their rate of addition was only about one-sixth that for internal hydrogen bonds amongst the protein atoms. The preferred residue exchanges and surface atom types useful in engineering enhanced stability were also examined.

Introduction

Why do some organisms survive temperatures above 100°C while others cease to live at 40°C? Why are some proteins extremely thermally stable while others denature at relatively low temperatures? Though such queries have prompted theoretical and experimental research for many years, the phenomenon of protein thermostability has been only partially understood despite its great importance in both the scientific and industrial arenas. Understanding the physicochemical principles of thermal stability will, no doubt, aid in the comprehension of protein folding and protein interaction mechanisms. Chemical reactions proceed with much higher speed if the reaction temperature is increased, yielding higher productivity as well as improved removal of unwanted reaction products destroyed by higher temperature.

Theoretical and experimental approaches have been undertaken to examine the stability of proteins (for reviews, see Gupta 1993, Russell and Taylor 1995, Querol et al 1996, Vieille and Zeikus 1996). Comparison of the sequences and tertiary structures of homologous proteins from thermophiles, mesophiles and thermophobes has formed the basis of theoretical efforts (e.g. Perutz and Raidt 1975, Argos et al 1979). Protein engineering, usually performed through site-directed mutagenesis, is the favorite mode of experimental analysis and stability enhancement Fersht and Serrano 1993, Matthews et al 1987. Querol et al. (1996) list at least 13 different physical and chemical reasons that researchers have reported in order to explain enhanced thermostabilization; these and other reasons are listed in Table 1. Though it may well be that proteins can be engineered or engineer themselves in vivo to achieve greater stability by utilizing one or more of these strategies, it is clear that no single and preferred mode has yet been found.

In this theoretical work, attempts were undertaken to find a largely consistent and straightforward explanation. First, all possible protein families, whose sequence-homologous members had known three-dimensional structure and optimal stability in natural environments of different temperature, were extracted from current data-bases. The resulting 16 families, the largest sample gathered to date, were examined for correlations between temperature of stability and polar surface fraction, as well as the number of hydrogen bonds and salt links between main and side-chain atoms. In analyses, 13 of the 16 families (over 80%) yielded a consistent pattern; namely, an increased temperature of stabilization was related to an increase in the number of hydrogen bonds amongst protein atoms and an increase in fractional polar atom exposed surface able to provide more hydrogen bonds with solvent. The number of salt bridges followed similar trends with increases in two-thirds of the families though they displayed only about one-sixth of the rate of increase found for hydrogen bonds. It is also noteworthy that in four recent studies Hennig et al 1995, Korndorfer et al 1995, Yip et al 1995, Salminen et al 1996 involving the experimental determination of the tertiary structures of particular hyperthermophilic proteins the researchers suggested an increase in ion pairs and/or hydrogen bonds as principal determinants of the increased stability. However, investigations of many thermophilic structures have led scientists to a myriad of explanations (Querol et al., 1996). The results reported here also point to the general significance of hydrogen bonds and ion pairs in the stability of proteins.

Section snippets

Choice of protein families

The three-dimensional protein structures used in this work were chosen from the Protein Data Bank (PDB) atomic coordinate database (Bernstein et al., 1977). The text for all proteins listed in PDB in October 1996 was searched for the word “thermo”. All entries matching this criterion were then examined manually for evidence that the associated protein was from a thermostable species. In a second step, the PDB text of all remaining entries was searched again for proteins from species previously

Solvation energy and exposed polar surface

Table 3 shows the change in solvation free energy upon folding (ΔΔG) amongst the tested families. In 11 of 16 families, the solvation free energy decreases to a greater extent with increasing thermostability.

Table 4 shows the actual compositional changes in fractional solvent exposed molecular surface for all the families and for various atom types. The net fractional surface area of the apolar carbon and sulfur atoms decreases in 13 of the 16 families while the net accessible surface area

Discussion

The number of experimentally determined three-dimensional structures of thermostable proteins is still relatively small and does not allow extensive statistical surveys, albeit in the present work the largest number of families have been examined which represents nearly three times those used in the earlier efforts of Menendez-Arias & Argos (1989). Making a protein more rigid may increase its thermostability but may also destroy its functionality. Since the functional requirements are very

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