Known functional effects of mutating hypothesized charged residues

Values are straight multiplications of WT affinities, so that 25 x WT is weaker binding and ½ WT is tighter binding.

Bandyopadhyay, P. K. and C.-W. Wu (1979). "Heterogeneity of the Two Tryptophanyl Residues on the lac Repressor of Escherichia coli." Arch.Biochem.Biophys. 195, No. 2: 558-564.

Barry, J. K. and K. S. Matthews (1997). "Ligand-induced conformational changes in lactose repressor: a fluorescence study of single tryptophan mutants." Biochemistry 36(50): 15632-42.

Barry, J. K. and K. S. Matthews (1999). "Substitutions at histidine 74 and aspartate 278 alter ligand binding and allostery in lactose repressor protein." Biochemistry 38(12): 3579-90.

Burns, L. E., A. H. Maki, et al. (1992). "Characterization of the two tryptophan residues of the lactose repressor from Escherichia coliby phosphorescence and optical detection of magnetic resonance." Biochemistry 32: 12821-12829.

Chakerian, A. E. and K. S. Matthews (1991). "Characterization of mutations in oligomerization domain of Lac repressor protein." J Biol Chem 266(33): 22206-14.

Chakerian, A. E., M. Pfahl, et al. (1985). "A mutant lactose repressor with altered inducer and operator binding parameters." J Mol Biol 183(1): 43-51.

Chang, W.-I., P. Barrera, et al. (1994). "Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: Aspartate 274 is essential for inducer binding in lac repressor." Biochemistry 33: 3607-3616.

Chang, W. I., J. S. Olson, et al. (1993). "Lysine 84 is at the subunit interface of lac repressor protein." J Biol Chem 268(23): 17613-22.

Chou, W. Y. and K. S. Matthews (1989). "Mutation in hinge region of lactose repressor protein alters physical and functional properties." J Biol Chem 264(11): 6171-6.

Dong, F., S. Spott, et al. (1999). "Dimerisation mutants of Lac repressor. I. A monomeric mutant, L251A, that binds Lac operator DNA as a dimer." J Mol Biol 290(3): 653-66.

Falcon, C. M. and K. S. Matthews (1999). "Glycine insertion in the hinge region of lactose repressor protein alters DNA binding." J Biol Chem 274(43): 30849-57.

Falcon, C. M. and K. S. Matthews (2000). "Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein." Biochemistry 39(36): 11074-83.

Falcon, C. M. and K. S. Matthews (2001). "Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery." Biochemistry 40(51): 15650-9.

Falcon, C. M., L. Swint-Kruse, et al. (1997). "Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage." J Biol Chem 272(43): 26818-21.

Gardner, J. A. and K. S. Matthews (1990). "Characterization of two mutant lactose repressor proteins containing single tryptophans." J Biol Chem 265(34): 21061-7.

Li, L. and K. S. Matthews (1995). "Characterization of Mutants Affecting the KRK Sequence in the Carboxyl-terminal Domain of lac Repressor." J.Biol.Chem. 270,no.18: 10640-10649.

M¸ller-Hartman, H. and B. M¸ller-Hill (1996). "The Side-chain of the Amino Acid Residue in Position 110 of the Lac Repressor Influences its Allosteric Equilibrium." J.Mol.Biol. 257,no.3: 473-478.

Ozarowski, A., J. K. Barry, et al. (1999). "Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants." Biochemistry 38(21): 6715-22.

Royer, C. A., J. A. Gardner, et al. (1990). "Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants." Biophys J 58: 363-378.

Spotts, R. O., A. E. Chakerian, et al. (1991). "Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins." J Biol Chem 266(34): 22998-3002.

Suckow, J., P. Markiewicz, et al. (1996). "Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure." J Mol Biol 261(4): 509-23.

Swint-Kruse, L., H. Zhan, et al. (2003). "Perturbation from a distance: mutations that alter LacI function through long-range effects." Biochemistry 42(47): 14004-16.

Swint-Kruse, L., H. Zhan, et al. (2005). "Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function." Biochemistry 44(33): 11201-13.

Zhan, H., L. Swint-Kruse, et al. (2006). "Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix." Biochemistry 45(18): 5896-5906.

Footnotes

[1] I^-: no repression; I^s, no induction. (Suckow, Markiewicz et al. 1996)

[2] (Barry and Matthews 1997)

[3] All other mutations at position 52: (Zhan, Swint-Kruse et al. 2006)

[4] C-red and Cox: (Falcon, Swint-Kruse et al. 1997; Falcon and Matthews 2001)

[5] Falcon Thesis

[6] (Falcon and Matthews 1999; Falcon and Matthews 2000)

[7] (Barry and Matthews 1999)

[8] Double mutant H74D/D278H; repeated below in D278 data.

[9] (Chou and Matthews 1989)

[10] (Chakerian, Pfahl et al. 1985)

[11] R, E, A, L: (Chang, Olson et al. 1993)

[12] A, L: (Swint-Kruse, Zhan et al. 2005)

[13] (Chang, Barrera et al. 1994)

[14] S. Dunning undergraduate honors project

[15] (M¸ller-Hartman and M¸ller-Hill 1996)

[16] (Swint-Kruse, Zhan et al. 2003)

[17] (Spotts, Chakerian et al. 1991)

[18] (Bandyopadhyay and Wu 1979; Gardner and Matthews 1990; Royer, Gardner et al. 1990; Burns, Maki et al. 1992; Barry and Matthews 1997; Ozarowski, Barry et al. 1999)

[19] (Dong, Spott et al. 1999)

[20] (Chakerian and Matthews 1991)

[21] (Li and Matthews 1995)

	Phenotype[1]	Variant	O¹DNA	DNA + IPTG	IPTG pH 7.4	IPTG pH 9.2	IPTG +DNA	IPTG kinetics	Allostery	DNA Release
WT			~10^-11M	~ 10^-7M or 10,000 x DNA	~10^-6 M	~10^-5 M or 10 x pH 7.4	~10^-5 M or 10 x pH 7.4
Y7		W/Wless [2]
V52		A[3] C-red[4] C-ox D E F G H L P Q R S T W
Q55		E[5]	Very low
G58		+G [6]
Q60		G6 P G+G G+2G G+3G
L62		W/Wless[2]
H74	Some s	A [7] L D F W W/Wless[2] D/H278[8]	25 x WT 10 x WT 2 x WT ~₁/³WT ~₁/³WT 2 x WT	¾ WT₅/⁸ WT WT ½ x WT 12 x DNA ¼ x WT	~WT ~WT ~WT 10 x WT ~WT	= pH 7.4 = pH 7.4 = pH 7.4 7 x pH 7.4 = pH 7.4	2 x pH 7.4 2 x pH 7.4 4 x pH 7.4 14 x pH 7.4 2 x pH 7.4		IPTG bias IPTG bias DNA bias IPTG bias
S77	Weak – P is ws	L[9]	nonspecific		Biphasic WT/10^-5	~ pH 7.4
A81	- or s	V[10]	nonspecific	WT (nonspecific)	½ WT			Slower
K84	Mostly s R is + E is ws	R[11] E A L	WT WT WT 4 x WT	7 x DNA [12] 5 x DNA	10 x WT WT WT WT	2 x pH 7.4 = pH 7.4~ pH 7.4 2 x pH 7.4		Slow/ Biphasic Slow/ biphasic	Reduced[12] Reduced	WT and 2^ndIPTG[12] WT and 2^ndIPTG
D88	All s	A [13] E K N
E100[14]	+	L W/Wless[2]	WT		~WT or tighter	= pH 7.4 ↑ coop'tvty	Tighter than WT		Reduced?
A110		T[15] K	weaker? weaker?		~WT 100 x WT
Q117		W/Wless[2]
R118	Weak -, K and E are -
D130		A[13] E K N
L148		F [16]
D149	K is -, E and R are s, many other s
S151		P[16]
R197	All s	G[17] L K
W201 [18]
D219	+
W220[18]
F226		W/Wless[2]
D247	-, His is +sh
L251		A[19]
Y273		W/Wless[2]
D274	most are s E is + K, R are -	A[13] E K N
E277	+
D278	- or s E is s K,R –	A[7] L N H E K H/D74	~₁/³ WT ~₁/³ WT ~₁/³ WT ~WT ~₁/³WT ½ WT 2 x WT	2 x WT ½ WT ¼ WT 5 x WT 4 x WT ¼ x WT	6 x WT 6 x WT 2 x WT 2 x WT 2 x WT ~WT	5 x pH 7.4 5 x pH 7.4 2 x pH 7.4 4 x pH 7.4 4 x pH 7.4 = pH 7.4	15 x pH 7.4 10 x pH 7.4 12 x pH 7.4 9 x pH 7.4 8 x pH 7.4 2 x pH 7.4		DNA bias DNA bias DNA bias DNA bias DNA bias IPTG bias
C281[20]		S A F M I s/282D
Y282		F A L
F293		W/Wless[2]
P320		A[16]
K325		W/Wless[2]
R326[21]		K A E L W