Mutagenesis-based definitions and probes of residue burial in proteins -- Bajaj et al. 102 (45): 16221 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Bajaj et al. 10.1073/pnas.0505089102.

Supporting Information

Files in this Data Supplement:

Supporting Table 5
Supporting Table 6
Supporting Table 7
Supporting Figure 2
Supporting Table 8
Supporting Table 9
Supporting Figure 3
Supporting Figure 4
Supporting Figure 5
Supporting Figure 6

Supporting Figure 2

Fig. 2. Solubilities of Asp mutants of CcdB at buried positions in Escherichia coli strain CSH501. Lane 1, uninduced WT CcdB culture; lane 2, WT CcdB whole-cell lysate after induction with arabinose; lane 3, supernatant of WT CcdB after lysis; lane 4, pellet of WT CcdB after lysis; lane 5, pellet of 5D mutant after lysis; lane 6, supernatant of 5D mutant after lysis; lane 7, pellet of 17D after lysis; lane 8, supernatant of 17D after lysis; lane 9, CcdB marker.

Supporting Figure 3

Fig. 3. Expression and solubility of WT maltose-binding proteing (MBP) and its mutants in Pop6590. Lane 1, uninduced WT MBP culture; lane 2, WT MBP whole-cell lysate after induction with arabinose; lane 3, WT supernatant after lysis; lane 4, WT pellet after lysis; lane 5, MBP 224D whole-cell lysate after induction with arabinose; lane 6, MBP 264D whole-cell lysate after induction with arabinose; lane 7, MBP marker.

Supporting Figure 4

Fig. 4. Expression and solubility of WT thioredoxin (Trx) and its mutants in A307. Lane 1, uninduced WT Trx culture; lane 2, WT Trx whole-cell lysate after induction with arabinose; lane 3,Trx 25D whole-cell lysate after induction with arabinose; lane 4, Trx 78D whole-cell lysate after induction with arabinose; lane 5, Trx marker.

Supporting Figure 5

Fig. 5. Spectral characteristics of WT Trx and Trx 78D. (A) Far-UV CD spectra of WT (solid line) and Trx 78D (dashed line) in phosphate buffer (pH 7.0). (B) Near-UV CD spectra of WT Trx (solid line) and Trx 78D (dashed line) in phosphate buffer (pH 7.0). (C) Fluorescence emission spectra of WT Trx in absence of DTT (solid line), 78D Trx in absence of DTT (dotted line), WT in presence of DTT (dashed line), and 78D in presence of DTT (triangles).

Supporting Figure 6

Fig. 6. Thioredoxin activity assayed by catalysis of insulin aggregation. Aggregation of insulin was monitored by the intensity of scattered light at 650 nm in the presence of WT-Trx and DTT (─), 78DTrx and DTT (…), and only DTT (---).

Table 5. Phenotype of CcdB mutants in the absence of arabinose (0%)

Residue	Ala	Asp	Glu	Lys	Arg	ACC, %	Depth, Å	Secondary structure*
1M	I	I	I	I	I	77	4.6	O
2Q	I	I	I		A	17	4.7	O
3F	I		A	I	I	12	5.5	O
4K			I	WT	I	31	6.0	E
5V	I	I	I		A	0	7.5	E
6Y	I	I	I	I		9	6.2	E
7T				I		34	5.0	E
8Y	A	A	A		A	11	5.2	E
9K	A	A	A	WT		64	3.7	O
10R	A	A	A	A	WT	77	3.8	O
11E	A	A	WT		A	111	3.6	O
12S	A	A	A	A		43	4.0	O
13R	A	A	A	A	WT	84	3.5	O
14Y	A		I			17	5.1	O
15R	A	A	A	A	WT	74	4.0	O
16L	A	I	I	I		11	6.0	E
17F	A	I	I		I	0.2	8.4	E
18V		I	I	I	I	0.0	10.1	E
19D	A	WT		I	I	1.5	7.9	E
20A	A		I	I	I	0.0	5.5	O
21Q		I	I	I	I	1.1	4.7	O
22S	A	I	I	I	I	9	4.3	O
23D	A	WT	A	A	A	60	3.9	O
24I	I	I	I	I		66	3.7	O
25I	A	I	A			15	4.1	O
26D	A	WT	A	A	A	92	3.6	O
27T	A	A	A	A	A	10	4.2	O
28P	A		A	A	A	80	3.6	O
29G	A	A	A	A	A	66	3.8	O
30R	A		A	A	WT	46	4.3	E
31R	A	A			WT	17	5.4	E
32M	A	I	I	I	I	0.4	4.9	E
33V		I	I	I	I	1.3	7.2	E
34I	I	I	I	I		0	8.1	E
35P	I	I		I	I	9	6.6	E
36L	I	I	I	I	I	0	8.6	E
37A	WT	A	A		A	12	6.0	E
38S	A	A		A	A	40	4.7	E
39A	WT		A	A	A	14	4.9	O
40R		A		A	WT	80	3.6	O
41L	A	A	A	I		99	3.6	O
42L	A		A	A	A	37	4.3	O
43S	A		A	A	A	67	4.1	O
44D	A	WT	A	A	A	102	3.7	O
45K	A	A	A	WT		93	3.5	O
46V	A	A	A	A	A	49	4.3	O
47S	A	A	A	I	A	58	4.2	O
48R	A	A	A	A	WT	68	4.3	O
49E	A	A	WT	A	A	76	4.3	O
50L	A	I	I	I	A	11	5.2	O
51Y		I	A	A	A	17	5.6	O
52P	I	I			A	3	6.6	O
53V		A		A	A	26	4.5	E
54V	A	I	I	I	I	0.1	6.8	E
55H	A	I	A	A	A	66	3.9	E
56I	A	I	I	I	I	13	4.9	E
57G		A	A	A	A	124	3.7	O
58D		WT	A	A	A	119	3.5	O
59E	A		WT		A	36	4.4	E
60S		A	A	A	A	38	4.9	E
61W	A	A		A	A	13	6.1	E
62R	I	A	A	A	WT	7	6.3	E
63M	A	I	I	I	I	0.1	9.3	E
64M	A	A	A	A	A	22	6.4	O
65T	A		I			0.0	7.1	O
66T	A	A		A	A	13	4.0	O
67D	A	WT	A		A	34	4.1	O
68M	A		I	I	I	0.0	4.4	O
69A	WT	A		A		45	4.3	E
70S	A	A	A	A	A	7	4.1	E
71V	A	A	A	A	A	14	5.6	E
72P	A	A		I	A	36	4.7	E
73V		A		A	A	60	4.1	O
74S	A	A	A	A	A	86	3.8	O
75V	A			I	A	7	5.6	O
76I	A	A	A	A	A	17	4.9	E
77G	I	A	A	A	A	7	4.5	E
78E	A	A	WT	A	I	91	3.9	E
79E	A	A	WT	A	A	43	3.8	E
80V	A	I	A	A	A	36	3.9	E
81A	WT	A		A	A	33	4.3	E
82D		WT		A	A	48	4.0	E
83L		I	A	A	I	1.2	6.7	O
84S	A	A	A	A	A	60	4.2	O
85H	A	A	A	A	A	67	3.7	O
86R		A	A	A	WT	25	4.8	O
87E	A	A	WT	I	I	47	4.4	H
88N	A	A	A	A	I	88	4.1	H
89D	A	WT	A	A		51	4.9	H
90I	I	I	I	I	I	0.1	7.1	H
91K	A	A	I	WT		42	4.4	H
92N	A		A	A	A	64	4.3	H
93A	WT	I	I	I	I	0	6.9	H
94I	I	I	I	I	A	0.6	5.3	H
95N	A	I	I	I		39	4.4	H
96L		I	A	A	I	35	4.3	H
97M	I	I	I	I	I	0	4.2	H
98F	I	I	I	I	I	0.8	4.0	H
99Y			I	I	I	19	3.7	H
100G	I	I	I	I		3	4.0	O
101I	I	I	I	I		125	3.4	O

Blank spaces indicate that the mutant could not be constructed. A and I refer to active and inactive phenotypes, respectively.

*O, H, and E in the secondary structure column refer to residues in nonstructured, helix, and b-strand regions of CcdB, respectively.

Table 6. Phenotype of CcdB mutants at 0.1% arabinose

Residue	Ala	Asp	Glu	Lys	Arg	ACC, %	Depth	Secondary structure*
1M	I	I	I	I	I	77	4.6	O
2Q	A	I	I		A	17	4.7	O
3F	A		A	A	A	12	5.5	O
4K			I	WT	I	31	6.0	E
5V	A	I	I		A	0	7.5	E
6Y	A	I	A	A		9	6.2	E
7T				A		34	5.0	E
8Y	A	A	A		A	11	5.2	E
9K	A	A	A	WT		64	3.7	O
10R	A	A	A	A	WT	77	3.8	O
11E	A	A	WT		A	111	3.6	O
12S	A	A	A	A		43	4.0	O
13R	A	A	A	A	WT	84	3.5	O
14Y	A		I			17	5.1	O
15R	A	A	A	A	WT	74	4.0	O
16L	A	A	A	I		11	6.0	E
17F	A	I	A		A	0.2	8.4	E
18V		I	I	I	I	0.0	10.1	E
19D	A	WT		A	A	1.5	7.9	E
20A	A		I	I	I	0.0	5.5	O
21Q		I	A	A	A	1.1	4.7	O
22S	A	A	A	A	A	9	4.3	O
23D	A	WT	A	A	A	60	3.9	O
24I	I	I	I	I		66	3.7	O
25I	A	I	A			15	4.1	O
26D	A	WT	A	A	A	92	3.6	O
27T	A	A	A	A	A	10	4.2	O
28P	A		A	A	A	80	3.6	O
29G	A	A	A	A	A	66	3.8	O
30R	A		A	A	WT	46	4.3	E
31R	A	A			WT	17	5.4	E
32M	A	I	A	A	A	0.4	4.9	E
33V		I	A	A	A	1.3	7.2	E
34I	A	I	I	I		0	8.1	E
35P	A	A		A	I	9	6.6	E
36L	A	I	I	I	I	0	8.6	E
37A	WT	A	A		A	12	6.0	E
38S	A	A		A	A	40	4.7	E
39A	WT		A	A	A	14	4.9	O
40R		A		A	WT	80	3.6	O
41L	A	A	A	I		99	3.6	O
42L	A		A	A	A	37	4.3	O
43S	A		A	A	A	67	4.1	O
44D	A	WT	A	A	A	102	3.7	O
45K	A	A	A	WT		93	3.5	O
46V	A	A	A	A	A	49	4.3	O
47S	A	A	A	I	A	58	4.2	O
48R	A	A	A	A	WT	68	4.3	O
49E	A	A	WT	A	A	76	4.3	O
50L	A	A	A	A	A	11	5.2	O
51Y		I	A	A	A	17	5.6	O
52P	A	I			A	3	6.6	O
53V		A		A	A	26	4.5	E
54V	A	I	A	A	A	0.1	6.8	E
55H	A	I	A	A	A	66	3.9	E
56I	A	A	A	A	I	13	4.9	E
57G		A	A	A	A	124	3.7	O
58D		WT	A	A	A	119	3.5	O
59E	A		WT		A	36	4.4	E
60S		A	A	A	A	38	4.9	E
61W	A	A		A	A	13	6.1	E
62R	A	A	A	A	WT	7	6.3	E
63M	A	I	I	I	I	0.1	9.3	E
64M	A	A	A	A	A	22	6.4	O
65T	A		I			0.0	7.1	O
66T	A	A		A	A	13	4.0	O
67D	A	WT	A		A	34	4.1	O
68M	A		I	A	A	0.0	4.4	O
69A	WT	A		A		45	4.3	E
70S	A	A	A	A	A	7	4.1	E
71V	A	A	A	A	A	14	5.6	E
72P	A	A		A	A	36	4.7	E
73V		A		A	A	60	4.1	O
74S	A	A	A	A	A	86	3.8	O
75V	A			A	A	7	5.6	O
76I	A	A	A	A	A	17	4.9	E
77G	A	A	A	A	A	7	4.5	E
78E	A	A	WT	A	A	91	3.9	E
79E	A	A	WT	A	A	43	3.8	E
80V	A	A	A	A	A	36	3.9	E
81A	WT	A		A	A	33	4.3	E
82D		WT		A	A	48	4.0	E
83L		I	A	A	A	1.2	6.7	O
84S	A	A	A	A	A	60	4.2	O
85H	A	A	A	A	A	67	3.7	O
86R		A	A	A	WT	25	4.8	O
87E	A	A	WT	A	A	47	4.4	H
88N	A	A	A	A	A	88	4.1	H
89D	A	WT	A	A		51	4.9	H
90I	A	I	I	I	I	0.1	7.1	H
91K	A	A	I	WT		42	4.4	H
92N	A		A	A	A	64	4.3	H
93A	WT	I	I	A	A	0	6.9	H
94I	A	I	I	A	A	0.6	5.3	H
95N	A	I	A	A		39	4.4	H
96L		A	A	A	A	35	4.3	H
97M	A	I	A	A	A	0	4.2	H
98F	I	I	I	A	I	0.8	4.0	H
99Y			I	I	I	19	3.7	H
100G	I	I	I	I		3	4.0	O
101I	I	I	I	I		125	3.4	O

Blank spaces indicate that the mutant could not be constructed. A and I refer to active and inactive phenotypes, respectively.

*O, H, and E refer to residues in nonstructured, helix, and b-strand regions of CcdB, respectively.

Table 7. Level of tolerance with different substitutions in absence and presence of arabinose

Substitution	Active, %
Substitution	0% arabinose	0.1% arabinose
Alanine	77	93
Aspartate	55	64
Glutamate	58	73
Lysine	58	83
Arginine	62	84

Table 8. Z-scores that represent the significance of correlation of ACC from various homology-modeled structures of CcdB and mutagenesis data

Template	Total score		Z score		*rmsd, Å**
Template	All	Hydr	All	Hydr	*rmsd, Å**
CcdB	70	29	2.7	2.6	0.3
1ne8 (DALI)	64	25	1.9	1.7	2.7
1m1f (DALI)	61	26	1.5	2.0	3.4
1ub4 (DALI)	63	23	1.8	1.2	3.3
1m4z (DALI)	51	16	0.08	-0.4	8.0
1ne8 (3DPSSM)	46	19	-0.6	0.3	6.8
1m1f (FUGUE)	45	14	-0.8	-0.9	8.2
1ne8 (FUGUE)	51	19	0.08	0.3	7.2
1ub4 (3DPSSM)	51	18	0.08	0.03	8.1
1ub4(FUGUE)	51	20	0.4	0.5	13.9
1qfj (3DPSSM)	49	18	-0.2	0.03	18.5
1fhx (3DPSSM)	47	16	-0.5	-0.4	19.0
1pdo (123D)	47	15	-0.5	-0.7	16.6
1aqt (123D)	45	16	-0.8	-0.4	19.4
1f7d (123D)	43	17	-1.0	-0.2	17.3
1doq (FUGUE)	43	12	-1.0	-1.4	17.4
1sf9 (FUGUE)	50	16	-0.1	-0.4	18.6
3ezm (FUGUE)	44	15	-0.9	-0.7	20.9
3wrp (FUGUE)	45	12	-0.8	-1.4	19.3
1d9c (FUGUE)	50	16	-0.1	-0.4	19.9
1btn (FUGUE)	47	16	-0.5	-0.4	16.7
1lgp (FUGUE)	44	14	-0.9	-0.9	20.2
2trx (FUGUE)	51	19	0.1	0.3	12.7

Each homology model was derived from the indicated template (Protein Data Bank ID code) using alignments generated by the program in parentheses. "All" refers to the total score calculated using data from all 75 Asp mutants generated in this study. "Hydr" refers to the total score calculated for Asp mutants of hydrophobic amino acids only (35 mutants).

*rms deviation (rmsd) of homology model with the crystal structure (Protein Data Bank ID code 3vub) of CcdB.

Table 9. Z-scores that represent the significance of correlation of ACC between various homology models and the x-ray crystallographic structure of CcdB

Template	Total score		Z -score		*rmsd, Å**
Template	All	Hydr	All	Hydr	*rmsd, Å**
CcdB	101	45	2.9	2.8	0.3
1ne8 (DALI)	82	35	1.6	1.5	2.7
1m1f (DALI)	82	37	1.6	1.8	3.4
1ub4 (DALI)	81	33	1.5	1.3	3.3
1m4z (DALI)	69	24	0.4	0.1	8.0
1ne8 (3DPSSM)	58	27	-0.3	0.5	6.8
1m1f (FUGUE)	60	21	-0.1	-0.3	8.2
1ne8 (FUGUE)	67	28	0.4	0.6	7.2
1ub4 (3DPSSM)	63	24	0.1	0.1	8.1
1ub4(FUGUE)	66	24	-0.4	0.1	13.9
1qfj (3DPSSM)	55	21	-0.5	-0.3	18.5
1fhx (3DPSSM)	54	20	-0.6	-0.4	19.0
1pdo (123D)	50	15	-0.9	-1.1	16.6
1aqt (123D)	58	21	-0.3	-0.3	19.4
1f7d(123D)	50	16	-0.9	-0.9	17.3
1doq (PQS)	59	18	-0.2	-0.7	17.4
1sf9 (PQS)	58	16	-0.3	-0.9	18.6
3ezm (PQS)	53	20	-0.7	-0.4	20.9
3wrp (PQS)	51	14	-0.8	-1.2	19.3
1d9c (PQS)	57	20	-0.3	-0.4	19.9
1btn (PQS)	54	19	-0.6	-0.5	16.7
1lgp (PQS)	45	15	-1.3	-1.1	20.2
2trx (PQS)	53	19	-0.7	-0.5	12.7

Each homology model was derived from the indicated template (Protein Data Bank ID code) using alignments generated by the program in parentheses. "All" refers to the total score calculated using using ACC of all 101 residues of CcdB. "Hydr" refers to the total score calculated using hydrophobic amino acids only (50).

*rmsd of homology model with the crystal structure (Protein Data Bank ID code 3vub) of CcdB.