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%


Lin, Mol Vis 1997; 3:17 <http://www.emory.edu/molvis/v3/lin>
©1997 Molecular Vision
ISSN 1090-0535