Biotechnology Letters 26: 1137–1140, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 1137 Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle Nevin Gül Karagüler 1,∗ , Richard B. Sessions, Kathleen M. Moreton, Anthony R. Clarke & J. John Holbrook † Department of Biochemistry, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK 1 Current address: Istanbul Technical University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, Istanbul, Turkey ∗ Author for correspondence (Fax: +90-212-2856386; E-mail: karaguler@itu.edu.tr) Received 19 March 2004; Revisions requested 5 April 2004; Revisions received 10 May 2004; Accepted 10 May 2004 Key words: double mutant cycle, formate dehydrogenase, hydrogen bonding, modelling Abstract An homology model of Candida methylica formate dehydrogenase (cmFDH) was constructed based on the Pseudo- monas sp. 101 formate dehydrogenase (psFDH) structure. In wild type cmFDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by −4 kcal mol −1 . Introduction Various forces stabilize folded proteins (Lazaridis et al. 1995). Although there is still uncertainty about the energetic contribution of hydrogen bonding to pro- tein stability, hydrogen bonding is an important feature in the structure of proteins. The energetic magnitude of a hydrogen bond has been estimated at −3 to −9 kcal (Fersht 1999). The energetic values of hy- drogen bonds in native proteins and their denatured states are difficult to measure either directly or in- directly (Pace 1995). Byrne et al. (1995) estimated a range of energies from approx. −1.5 to −4 kcal mol −1 for the contribution of hydrogen bonding interactions to the stability of the native state by making a series of single substitution mutations in the model protein staphylococcal nuclease. Proteins have highly co-operative structures. Muta- tion at one residue may disrupt interactions with other residues. In many cases, interactions of several amino acid residues are coupled and may not be reduced † Deceased. to a sum of pair-wise interactions. Results of single mutation experiments are, for these reasons, prone to misinterpretation (Serrano et al. 1990). This problem may be at least partially overcome by invoking double mutant cycles (Carter et al. 1984) which enable the measurement of pair-wise interactions in proteins. The double mutant cycle method was applied to estimate the strength of the polar interaction between two hydrophilic residues, Thr169 and Thr226, on adja- cent parallel β -sheets of cmFDH. If the two threonines form a hydrogen bond with one another we would ex- pect changing just one of them to valine might make the protein less stable as this hydrogen bond would be broken. However, when both threonines are re- placed by valines, a larger hydrophobic core would be produced. This would either increase or decrease the stability of the protein. Hydrophilic threonines at po- sitions 169, 226 were mutated to hydrophobic valine residues separately and together.