Folding of circular permutants with decreased contact order 1 Folding of circular permutants with decreased contact order: general trend balanced by protein stability MAGNUS O. LINDBERG, JEANETTE T¯NGROT 1 , DANIEL E. OTZEN 2 , DMITRY A. DOLGIKH 3 , ALEXEI V. FINKELSTEIN 4 AND MIKAEL OLIVEBERG * Department of Biochemistry, Ume University, S-901 87 Ume, Sweden. 1 Department of Computing Science, Ume University. 2 Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. 3 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871, Moscow, Russia. 4 Institute of Protein Research, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia * To whom reprint requests should be addressed. E-mail: mikael.oliveberg@chem.umu.se . Summary To examine the influence of topology and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small β-α-β protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log k f ) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na 2 S0 4 or by comparison of the folding kinetics at the transition midpoint, the dependence between log k f and contact order becomes stronger and follows the general correlation for small β-α-β proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding. Key words: protein folding, rate constants, two-state proteins, topology, protein stability, transition state. Abbrevations: GdmCl, guanidinium chloride.