Table II.

Determination of the effect of engineering of the C-terminal AB-, CD- and EF-loops of the CH3 domains (Fig. 1) of IgG1-Fc on the structural fitness of the resulting libraries

AB-loop libraries (RDELTKNQ)	ΔT1/2 (°C)	CD-loop libraries (SNGQPENNY)	ΔT1/2 (°C)	EF-loop libraries (DKSRWQQGNV)	ΔT1/2 (°C)
LT	−2.8 ± 0.7	SN	−2.1 ± 0.8	QQGNV	−3.3 ± 0.1
LTK	−3.9 ± 0.5	NG	−2.8 ± 0.9	RWQQGNV	−5.0 ± 0.2
LTKN	−3.5 ± 0.7	GQ	−3.0 ± 0.6	DKSRWQQGNV	−8.0 ± 0.6
TK	−1.1 ± 0.4	QP	−1.8 ± 0.7	DK	−2.1 ± 0.3
TKN	−1.1 ± 0.7	PE	−2.9 ± 0.6	KS	−2.4 ± 0.2
KN	−1.7 ± 0.5	EN	−3.1 ± 0.9	DKS	−2.4 ± 0.3
RDELTKN	−9.7 ± 1.0	NN	−1.4 ± 0.8	DKSRW	−5.3 ± 0.3
		NY	−4.4 ± 0.7	RW	−3.6 ± 0.2
				DKSrwQQGNV	−6.3 ± 0.4
AB insertion libraries		CD insertion libraries		EF insertion libraries
LT5	−5.7 ± 0.9	SN5	−4.4 ± 0.4	DK5	−8.7 ± 0.5
LTK5	−6.0 ± 0.8	NG5	−3.7 ± 0.3	KS5	−8.3 ± 0.7
LTKN5	−5.2 ± 0.8	GQ5	−4.4 ± 0.2
TK5	−3.8 ± 0.7	QP5	−4.5 ± 0.1
TKN5	−4.0 ± 0.9	PE5	−5.4 ± 0.2
KN5	−3.9 ± 0.7	EN5	−5.3 ± 0.1
		NN5	−5.1 ± 0.1
		NY5	−7.0 ± 0.1

The randomized residues in the respective loops are indicated. Furthermore, insertion libraries with five additional residues inserted after the (also randomized) amino acid positions were designed. Constructed libraries were expressed on the surface of yeast and cell suspensions were incubated at increasing temperatures and (after cooling) were probed for binding to FcγRI using flow cytometry. Obtained ΔT_1/2 values (for calculation see Fig. 2 and the Materials and methods Section) describe the overall destabilization of a distinct library as a consequence of randomization with respect to the displayed wild-type protein.