Table 1.

Library design and binding parameters of affinity clamps

Protein	BC loop (25–30)*	DE loop (52–55)*	FG loop (75–83)*	kon,† M−1S−1 (ARVCF)	koff,† S−1 (ARVCF)	Kd, nM		IC50,† nM (ARVCF)	Affinity enhancement‡	Specificity index§
						ARVCF	δ-catenin
Library¶	X4–8 VX	(SY)(GSY) (S/Y)2	X8–14	—	—	—	—	—	—	—
PDZ	—	—	—	ND	ND	2,200	6,300	3,000‖	—	3
cpPDZFN	SSSSVS	GSKS	SSSSSSSSS	ND	ND	24,800	>25,000	ND	1	ND
ePDZ-a	SYYGVS	YSSS	YSDYYGSHHY	2.9 × 105	1.5 × 10−2	56 ± 5	429	40	520	8
ePDZ-b	YYDSHVS	GSKS	HYNYHYYS	1.9 × 105	1.1 × 10−2	56 ± 6	>25,000	59	520	>446
ePDZ-b1	YRELPVS	GSKS	HYNYHYYS	7.3 × 104	<3.7 × 10−4	<5	>25,000	ND	>4,960	>5,000
ePDZ-b2	FTDLPVS	GSKS	HYNYHYYS	7.0 × 104	<2.9 × 10−4	<4	>25,000	ND	>6,200	>6,250

ND, not determined.

*The residue numbering is according to that in Koide et al. (21).

^†The k_on and k_off are shown for the ARVCF peptide. IC₅₀ for the ARVCF peptide was determined by using competitive phage ELISA.

^‡The affinity enhancement is defined as the ratio of the K_d of the parent protein (cpPDZFN) for ARVCF binding to that of an affinity clamp.

^§The specificity index is defined as the ratio of the K_d for δ -catenin to the K_d for ARVCF.

^¶The combinatorial library was constructed by diversifying the BC, DE, and FG loops of FN3. X denotes an amino acid mixture consisting of 40% Tyr, 20% Ser, 10% Gly, and 5% each of A, D, H, L, N and R.

^‖Taken from Zhang et al. (42).