Biochemical and biophysical characterization of combinatorial mutants. (a) Examples of raw data for in vitro cleavage (see Materials and Methods). Different concentrations of proteins were assayed. Lanes 1 to 15: protein concentrations in nM are 250, 189.4, 126.3, 84.2, 63.2, 42.1, 21.1, 15.8, 10.5, 7.4, 4.2, 2.1, 1.0, 0.5 and 0. (b) Cleavage of COMB2 by combinatorial mutants. (c) Cleavage of COMB3 by combinatorial mutants. (d) Thermal denaturation of the same proteins measured by CD. The bold line corresponds to I-CreI N75, with a mid point denaturation temperature of 65°C. Other proteins: KNHQS/KEG (mid point denaturation temperature: 65.3°C), KNHQS/KAS (64.9°C), KEG (63.1°C),KNHQS (62.2°C), NNSRQ (61.2°C), KAS (61.2°C), KAS (61.2°C), ARR (57.3°C), ASR (57.1°C), NNSRK/ARR (55.8°C), NNSRK/ASR (55.8°C). For protein nomenclature, see .