Tables for
Lin, Mol Vis 3:17, 1997.
Table Ia. Protein Variant Terminology
Protein Definition WT Wild Type human IRBP expressed in insect cells R1 Repeat 1 with signal peptide attached expressed in insect cells R12- Repeat 1 and part of Repeat 2, as one polypeptide, expressed in insect cells R12+ Repeats 1 and 2 plus part of Repeat 3, as one polypeptide, expressed in insect cells R123 Repeats 1, 2 and 3, as one polypeptide, expressed in insect cells G719S Whole human IRBP with a point change at position 719 changing a Glycine to a Serine, expressed in insect cells R725C Whole human IRBP with a point substitution at position 725 changing an Arginine to a Cysteine, expressed in insect cells EcR1 Repeat 1, expressed in E. coli EcR2 Repeat 2, expressed in E. coli EcR3 Repeat 3, expressed in E. coli EcR4 Repeat 4, expressed in E. coli |
Table Ib. Oligonucleotide primers used in the construction of altered IRBPs
Primer Sequence Used in producing the (Location of primer identity with IRBP sequences) following Proteins Primer 1 GGATCAGAGTCTGGAGCGGCCGCGAATTCC R1, R12-, R12+, R123 (Forward) (4128-4157) Primer 2 GCTCTAGACTATCAACTAGTGAGCATGGCCAGGGCTTTC R1 (Reverse) (5215-5236) Primer 3 GCTCTAGACTATCAGCGCGCCTCGCCGTGATTGTCGATGAAG R12- (Reverse) (6064-6087) Primer 4 GCTCTAGACTATCTACGCGAGTGGAACACTAGCAAGCGGTGG R12+ (Reverse) (6361-6382) Primer 5 GCTCTAGACTATCAGTCCCAGGCCTTGCCTGTGGTGGCAC R123 (Reverse) (6972-6997) Primer 6 GGGCCCTGAGGCCGGCCCCGTGCAC R725C, G719S (Forward) (5664-5688) Primer 7 GTTCAGCCATGGCGTCAAAACACAGGTAGCCCAGC R725C (Reverse) (6499-6523) Primer 8 TTCAGCCATGGCGTCAAAACGCAGGTAGCCCAGCTGACTGGGCAGC G719S (Reverse) (6487-6532) Primer 9 CGGGATCCGGGCCCCACACACCTGTTCC EcR1 (Forward) (4325-4343) Primer 10 CGGGATTCCTTATCAGTGGTGGTGGTGGTGGTGGCGCAGAGTGAGGATGGCC EcR1 (Reverse) (5224-5242) Primer 11 GGAATTCCGCAGCGCCCTTCCAGGGG EcR2 (Forward) (5240-5258) Primer 12 GGAATTCTTATCAGTGGTGGTGGTGGTGGTGGTGGAACTCCAGCACTTCC EcR2 (Reverse) (6172-6154) Primer 13 CGGGATCCGCAAAGCCTGGGGGCCTTGG EcR3 (Forward) (6173-6191) Primer 14 GGGATCCTTATCAGTGGTGGTGGTGGTGGTGACGCAGAGCCACTATGTCC EcR3 (Reverse) (7075-7057) Primer 15 CGGGATCCGGCCAAGGTGCCCACGGTGC EcR4 (Forward) (7076-7096) Primer 16 CGGGATCCTTATCAGTGGTGGTGGTGGTGGTGCAGGTGGTCCTGCAGGCCTG EcR4 (Reverse) (8014-7995) |
Table Ic. Plasmid and Protein Construction Summary
Protein Clone Repeat Nucleot FWD Back Restric Poly Vector Name Name AAs ides Primer Primer Enzymes His AAs from tag? pVL4200 WT pVL4200 -22 to - - - - No -- 1225 R1 pR1 -22 to 4181 to 1 2 Not I, No -- 299 5236 Xba I R12- pR12- -22 to 4181 to 1 3 Not I, No -- 583 6088 Xba I R12+ pR12+ -22 to 4144 to 1 4 Not I, No See 678 6373 Not I text R123 pR123 -22 to 4181 to 1 5 Not I, No -- 886 6997 Xba I G719S pG719S -22 to 5778 to 6 7 Fse I, No -- 1225 6522 Nco I R725C pR725C -22 to 5778 to 6 8 Fse I, No -- 1225 6522 Nco I EcR1 pLexR1 -5 to 4325 to 9 10 BamH I, Yes 7 301 5242 BamH I EcR2 pLexR2 301 to 5240 to 11 12 EcoR I, Yes 17 611 6172 EcoR I EcR3 pLexR3 612 to 6173 to 13 14 BamH I, Yes 7 912 7075 BamH I EcR4 pLexR4 913 to 7076 to 15 16 BamH I, Yes 7 1225 8014 BamH I |
Table II. Secondary Structure Estimates based on CD Spectra
Protein --- Contin ---- ----- K2D ----- --------- Selcon ---------- ---------- Varslc ----------- alpha ß o alpha ß Rand alpha ßA ßP t o alpha ß A ß P t o WT 14 54 32 16 32 53 20 18 4 15 39 10 13 5 12 19 R1 12 46 42 15 32 53 13 29 1.4 20 33 17 0 0 1 19 EcR1 11 54 36 12 37 51 11 39 5 22 20 11 7 2 8 12 EcR2 19 49 32 15 30 55 30 0.6 7.7 21 37 17 5 0 9 12 EcR3 39 61 0 35 16 49 36 9 4.7 25 29 14 50 17 33 43 EcR4 9 50 40 16 32 52 14 27 23 21 32 14 0 0 5 10 R12- 6 58 36 12 40 48 9.3 7.8 3.5 8.2 14 7 1 0 3 5 R12+ 12 52 36 11 35 54 13 13 25 12 23 10 6 2 9 13 R123 21 39 41 25 19 56 35 6 5 24 26 21 0 0 8 11 G719S 22 51 27 19 28 53 22 16 3 21 35 16 14 4 15 23 R725C 29 47 24 26 25 49 25 15 30 21 31 25 20 6.1 20 28 |
Abbreviations: alpha, [alpha]-helix; ß, ß-strand; ßA, antiparallel ß-strand; ßP, parallel ß-strand; o, other; Rand, Random coil; t, turn
Table IIIa. Altered IRBP Proteins: Retinol ligand binding properties (Values ± SD)
Mutant/ Number of Fo S e Kd N Bmax (cps) variant trials (cps) (cps/µM) (A/µM/cm) (µM) for 100% pure protein (1 µM) WT+ 18 105 7840 0.0275 0.356 1.30 10200 (152) (3330) (0.0430) (0.239) (0.334) (3330) R123 7 49.2 7640 0.0936 0.0962 0.913 9960 (114) (1870) (0.0387) (0.0689) (0.244) (2670) R12+ 7 587 5290 0.0747 0.0275 0.730 5440 (115) (2140) (0.0566) (0.0220) (0.263) (3010) R12- 7 39.7 8540 0.123 0.0668 0.511 7520 (65.3) (3970) (0.170) (0.0921) (0.506) (6840) R1 7 98.2 5820 0.0642 0.0367 0.629 4360 (102) (1950) (0.0929) (0.0351) (0.0830) (2320) G719S 16 184 6080 -0.0122 0.125 0.923 5610 (138) (2780) (0.0391) (0.172) (0.335) (3660) R725C 11 116 6110 0.0237 0.163 1.47 8980 (43.2) (1860) (0.0379) (0.164) (0.483) (1860) EcR1 4 -2.01 6620 0.0499 0.386 1.31 8670 (11.6) (976) (0.0127) (0.418) (0.271) (976) EcR2 4 -29.8 3600 0.00638 0.0969 1.18 4250 (43.4) (356) (0.0120) (0.0601) (0.198) (356) EcR3 3 4.32 1490 -0.0380 0.00866 0.689 1030 (5.33) (528) (0.0351) (0.00861) (0.110) (528) EcR4 4 66.7 1960 0.0129 0.0363 1.20 2350 (20.4) (96.1) (0.0225) (0.0209) (0.221) (96.1) Median CV 123% 33.5% 145% 95.6% 25.7% 26.8% |
Table IIIb. Altered IRBP Proteins: 16-AP Ligand Binding Properties (Values ± SD)
Mutant/ Number of Fo S e Kd N Bmax (cps) variant trials (cps) (cps/µM) (A/µM/cm) (µM) for 100% pure protein (1 µM) WT+ 5 234 25700 0.546 0.0149 1.10 23400 (238) (9510) (0.477) (0.0170) (0.470) (9510) R123 4 218 26300 1.06 0.00620 0.716 26800 (64.3) (11400) (1.92) (0.00696) (0.744) (16300) R12+ 5 538 18000 -0.457 0.0232 0.564 14300 (172) (13900) (1.69) (0.0445) (0.734) (19600) R12- 4 117 15800 1.56 0.000461 0.507 13800 (144) (3080) (0.648) (0.000555) (0.0726) (17700) R1 4 341 33100 0.279 0.0376 0.246 9680 (478) (19000) (0.556) (0.0431) (0.104) (22600) G719S 4 32.5 28000 0.589 0.0223 0.817 30100 (56.8) (10500) (0.701) (0.0249) (0.381) (13800) R725C 4 7.41 24800 0.464 0.0262 0.641 15800 (108) (14300) (0.485) (0.0242) (0.282) (14300) EcR1 4 77.0 24100 0.948 0.00855 0.532 12800 (42.1) (3110) (2.56) (0.00892) (0.312) (3110) EcR2 6 10.8 33100 -0.00790 0.00510 0.263 8700 (28.7) (7050) (2.42) (0.00540) (0.161) (7050) EcR3 4 26.6 1580 -0.604 0.00440 0.309 488 (53.1) (247) (1.38) (0.00730) (0.230) (247) EcR4 5 54.4 4560 1.34 0.0111 0.250 1820 (31.0) (3210) (1.90) (0.0162) (0.0956) (3210) Median CV 123% 37.5% 181% 114% 46.6% 81% |