Table 2.

Peptide phage display and alanine scanning analysis of N-terminal V_L Len residues implicated in 11-1F4 binding.

VL Len residue position	Phage peptide		Len(1–18) Alanine scanning		Phage and Len(1–18) Residue Preferencesd
	Identitya(%)	Ala (%)	EC50b(nM)	EC50 WT EC50c
D1	3.8	0	3.91 ± 0.001	2.7	S or T
I2	15.4	19.2	0.27 ± 0.001	0.22	Ala or any amino acid
V3	9.6	0	0.29 ± 0.004	0.24	Polar
M4	15.4	0	2.51 ± 0.01	2.1	Aromatic (preferably Tyr)e
T5	82.7	10.0	1.48 ± 0.01	1.2	Small (preferably S or T)
Q6	5.8	4.0	2.33 ± 0.01	1.9	Polar
S7	26.9	8.0	0.62 ± 0.01	0.51	Polar (preferably aromatic)
P8	100	0	>500 ± 10	>400	Invariant proline
D9	61.5	2.0	43.2 ± 0.2	36	Negatively charged, D/E
S10	40.4	6.3	0.85 ± 0.003	0.71	Small polar (preferably S or T)
L11	80.8	0	77.1 ± 0.7	64	L or bulky hydrophobic
A12	5.8	0	2.86 ± 0.02d	2.4	Bulky hydrophobic
V13	73.0	2.6	18.6 ± 0.1	16	V or aliphatic
S14	30.7	0	7.96 ± 0.04	6.6	S or T
L15	n.a.f	n.a.	7.64 ± 0.02	6.4	(L)
G16	n.a.	n.a.	2.12 ± 0.01	1.8	n.a.
E17	n.a.	n.a.	2.48 ± 0.01	2.1	n.a.
R18	n.a.	n.a.	0.55 ± 0.002	0.5	n.a.

^aSequence identity for each phage peptide compared to V_L Len was determined as the sum of the percentages of identical and chemically similar (32) residues at each position from the multiple sequence alignments of phage peptide groups I and II shown in Figure 1.

^bEC₅₀ values were determined from the midpoint of 11-1F4 titration curves shown in Figure 6.

^cWT EC₅₀ is the EC₅₀ value for 11-1F4 binding to plate-immobilized wild-type Len(1–18).

^dResidue preference at each position in Len(1–18) was determined from the multiple sequence alignments of phage peptide groups I and II with VL Len, and the binding of 11-1F4 to Alasubstituted Len(1–18) peptides (Figure 1, Figure 2, and Figure 6). Residue preferences with or without brackets are those that were preferred for Len and phage peptides, respectively.

^eResidue preference in brackets are those specific to the Len(1–18) peptide.

^fn.a., not applicable.