HISTORIA DE LA PROTECCION POSESORIA

binding site of wild type NF-kB p50 aa35-381

Although the above evidence was very suggestive of the intimate association of cysteine 62 with the DNA binding site of p50, more direct evidence was needed for a definitive assignment. The approach chosen was of differential labelling - a method first used to radiolabel the active site of an antibody (Koshland et al.,

1959). Tlie rationale behind this choice was that since preformation of the DNA-protein complex could protect the cysteine residue(s) in the DNA binding site of p50 against iodoacetate inactivation, reaction of the DNA-protein complex

with unlabelled iodoacetate should modify all the exposed cysteine thiol(ate) groups. Then, after removal of the excess unlabelled iodoacetate, the DNA-protein complex could be dissociated by increasing the NaCl concentration to 0.6M and the newly-exposed cysteine groups radiolabelled by reaction with iodoacetate. An outline of the iodoacetate differential labelling scheme used is shown in Figure 5.3 - this used HIV enhancer oligonucleotide-Sepharose resin to bind the wild type NF-kB p50 aa35-381 protein during the reaction with unlabelled

iodoacetate, and allowed easy removal of the oligonucleotide binding site(s) from the reaction mixture.

The differential labelling (see Materials and Methods) of lOOug of wild type NF-kB p50 aa35-381 protein (Figure 5.4) illustrates some of the behaviour

seen before with wild type p50 protein - thus comparing the amount of «38kD

NF-kB p50 aa35-381 protein in the wash fractions (W1 and W2 - Figure 5.4,

Panel A), it can be seen that there is no evidence of iodoacetate treatment of the preformed DNA-protein complex causing breakdown of the complex. From the autoradiogram (Figure 5.4, Panel B) of the SDS-PAGE gel, it is clear there is only one radiolabelled species (albeit as a doublet band) in the track. It seems likely that the higher molecular weight polypeptide bands present in all tracks in Panel A represent some contaminant proteins, possibly present in the SDS gel sample buffer since from other gels there was no evidence of their presence in the p50 protein used as the starting material, nor would they be expected to be present in the C30FT Centricon-30 flowthrough fraction when there was no evidence of the ~38kD p50 protein in either the SDS-PAGE (Figure 5.4, Panel A) or gel electrophoresis DNA binding assays (Figure 5.4, Panel C).

Figure 5.3 Outline of iodoacetate labelling / substrate protection

scheme for cysteine residues in the DNA binding site of wild type NF-kB

p50 aa35-381 protein.

Given below is the outline of the DNA binding site substrate protection scheme used for lOOug of wild type p50 protein, the estimated recovery of radiolabelled wild type p50 for trypsin digestion was ~75ug.

100 Mg WT p50 / 20mM DTT (400^1) (L)

Add to 200^1 HlV-Sepharose resin, incubate 3 hours Centrifuge and remove supernatant (FT)

Wash resin with 2ml 0.1 M NaCl buffer / 2inM DTT (W l)

Add 250mM neutral iodoacetate to lOmM, incubate 15min at 20®C Wash rosin with 2ml O.IM NaCl buffer / 2mM DTT (W2)

Elute WT p50 with I ml 0.6M NaCl buffer / 2mM DTT Concentrate eluate in Centricon-30 down to ~30ppl, discard the filtrate (C30FT)

Add ^^C iodoacetate solution to ~IOmM, incubate 25min at 20*C Add DTT to 50mM to quench remaining iodoacetate

Precipitate protein by adding TCA to 10%

Rcdissolvc protein pellet in 50).il NH^IICO) buffer / 8M urea ^ Dilute with water down to 200pi / 2M urea

Digest with 3pg trypsin for 24hours at 3T C

Separate peptides by reverse phase HPLC on a CIS column with a 0 to 80% acetonitrile gradient, assay fractions for radioactivity

Figure 5.4 Analysis of fractions from the substrate protection

labelling of cysteine residues in the DNA binding site of wild type NF-kB

p50 aa35-381 protein.

A. SDS-PAGE analysis of substrate protection experiment (see Materials and

Methods) fractions, visualised by Coomassie staining:- M, protein molecular weight

standards; final p50 protein eluate after labeling; L, initial load (l(X)ug p50

protein, 400ul buffer) to HIV enhancer oligonucleotide-Sepharose affinity resin; FT, supernatant from loading p50 protein onto affinity resin; Wl, first O.IM NaCl wash of p50-Ioaded affinity resin; W2, second (after reaction of DNA-protein complex with unlabelled iodoacetate) O.IM NaCl wash of p50-loaded affinity resin; C30FT, discarded Centricon-30 filtrate from concentration of eluted p50 protein prior to reaction with iodoacetate. The following volumes of protein fractions were used for the SDS-PAGE

analysis - 8ul, L - 4ul, FT - 4ul, Wl - 20ul, W2 - 20ul, C30FT - 20ul. The

molecular weights (in kD) of the marker proteins are indicated.

B. Direct autoradiogram of the above SDS-PAGE dried gel, abbreviations as in Panel A. C. Gel electrophoresis DNA binding assays carried out under standard conditions (see Materials and Methods) in the presence of spermidine with ^ ^ P radiolabelled HIV-L kB

motif oligonucleotide. Abbreviations as used in Panel A, indicates no protein addition, 'L' and *FT* tracks used lul of protein solution, tracks 'Wl', 'W2', and 'C30FT' used 5ul of protein solution. B and F indicate the positions of the DNA-protein complex and free oligonucleotide bands respectively.

A C