Table 1.

Sequence characteristics and dissociation constants of the fluorescein-binding lipocalin variants

Residue no.	Residue present
	BBP	FluA	FluB	FluC	FluD
34	Asn	Ser	Gln	Ser	Glu
35	Ser	Pro	His	Lys	Ala
36	Val	Asn	Trp	Asn	Gly
37	Glu	Gly	Asp	Gly	Asn
58	Asn	Arg	Arg	Arg	Arg
60	His	Asp	Arg	Thr	Ser
69	Ile	Met	His	Gln*	Ser
88	Leu	Arg	Val	Arg	Ser
90	Tyr	Val	Arg	Val	Val
93	Val	Tyr	Arg	Lys	Arg
95	Lys	Arg	Arg	Arg	Arg
97	Asn	Thr	Gly	Gly	Val
114	Tyr	Ser	Arg	Arg	Arg
116	Lys	Arg	Arg	Arg	Leu
125	Gln	Trp	Trp	Leu	Arg
127	Phe	His	His	His	Leu
40	Gly			Arg†
68	Phe			Val†
70	Glu		Lys†
96	Glu	Lys†
Kd,‡ nM	>5·103¶	536 ± 174	55 ± 8	87 ± 11
Kd,§ nM	>105¶	427 ± 10	— ∥	61.8 ± 3.1

*Arising from an amber termination codon in a supE background. 

^†Accidental mutation position not included in the random mutagenesis. 

^‡Determined as the protein concentration giving rise to half-maximal binding in an ELISA with the immobilized fluorescein-RNase conjugate. 

^§Determined for 4-glutarylamidofluorescein by fluorescence titration of the protein. 

^¶Conservative estimate (cf. footnote to Table 2). 

^∥ Insufficient fluorescence quenching effect.