Structural characterization and biochemical analysis of ID2, an inhibitor of DNA binding 3

90% purity and greater was checked by SDS-PAGE Figure 9 and gel filtration elution profiles showed that the protein existed as dimers in solution Appendix 6.. Figure 8: ID2 proteins’ exp

(Figure 8, D, G, K) The Resource-S column, a strong cation exchanger, was chosen based on the fusion constructs’ pI and pH of buffer such that only ID2 bound to the column and not the tag At this stage, there was still some residual tag left To avoid protein loss, no gel filtration chromatography was performed Instead, a final round of reverse His-affinity chromatography (Figure 8E) was run to remove the residual tag Running at a slow gradient of increasing Imidazole concentration, the His6-containing tag was captured on the nickel beads while pure ID2 was eluted in the flowthrough (Figure 8, F, H) The seleno-methionine construct was pure enough after ion

exchange chromatography and did not require further purification The proteins were pooled and concentrated in buffer 50 mM TRIS-HCL pH8.0, 100mM NaCl after the final chromatography step 90% purity and greater was checked by SDS-PAGE (Figure 9) and gel filtration elution profiles showed that the protein existed as dimers

in solution (Appendix 6) These samples were then used for crystallization

experiments

Figure 8: ID2 proteins’ expression and purification

(A) Elution profile of nickel-bead affinity and desalting chromatography

(B) SDS-PAGE: marker (lane 1), sample before induction (lane 2), pooled desalted fractions (lane 3), TEV-cleaved affinity tag (lane 4)

(C) Elution profile of ion-exchange chromatography with increasing salt gradient

(D) SDS-PAGE: marker (lane 1), input sample after TEV (lane 2), flowthrough (lane 3), fractions

at positions of crosses in profile from Fig 3.1C (lane 4-11)

(E) Reverse affinity chromatography profile; ID2 eluted in flowthrough, residual tag bound to nickel beads

(F) SDS-PAGE: Representative ID2 fractions (lane 1-2), marker (lane 3), affinity tag (lane 4)

Figure 8 continued:

(G) N-HLH82-L expression and purification SDS-PAGE: marker (lane 1), pooled affinity and desalting chromatography fractions before TEV (lane 2), TEV cleaved (lane 3), unbound ion exchange fraction (lane 4), ion exchange elutions (lane 5-10)

(H) N-HLH-82-L reverse affinity chromatography SDS-PAGE: input sample (lane 1), marker (lane 2), bound tag (lane 3), unbound ID2 fractions (lane 4-12)

(J) HLH24-82-L-Se-Met SDS-PAGE: marker (lane 1), sample before induction (lane 2), pooled affinity and desalting fractions before TEV (lane 3), TEV cleaved (lane 4)

(K) HLH24-82-L-Se-Met ion exchange samples SDS-PAGE: input sample after TEV (lane 1), marker (lane 2), eluted bound ID2 fractions (lane 3-8)

Figure 9: ID2 proteins’ purity check by SDS_PAGE: marker (lane M, kDa) N-HLH82-L (gel A, lane 1), HLH24-82-L (gel B, lane 2), HLH24-82-L-Se-Met (gel C, lane 3)

3.3 Protein Identification

The purified samples were excised from the gel and analyzed by Liquid

chromatography tandem mass spectrometry (LC/MS/MS) for peptide mass

fingerprinting Searches were made both against all non-redundant proteins as well

as just the human dataset to show that no matter which dataset was used, the results were the same and the identity of the protein was that of ID2 (Table 7) The only difference was that the longer form of ID2, N-HLH82-L contained the intact N-terminal region of the protein as shown in matched peptides in bold red

Table 7: LC/MS/MS mass spectrometry top hits for the purified proteins (Figure 8) Searches were done against all nr as well as human nr to show that the fragments captured always belonged to ID2 Note that the N-HLH-82-L contained the intact N-terminus (matched peptides in bold red) whereas the shorter form HLH24-82-L and the seleno-methionine version did not

N-HLH82-L

(searched

against all

non-redundant

proteins)

Match to: IPI00294210 Score: 4673 Tax_Id=9606 Gene_Symbol=ID2 DNA-binding protein inhibitor ID-2

Found in search of C:\Documents and Settings\Administrator\Desktop\230609_p565C6_pure.RAW Nominal mass (M r): 15022; Calculated pI value: 7.82

NCBI BLAST search of IPI00294210 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C)

Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P

Sequence Coverage: 42%

Matched peptides shown in Bold Red

101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG

HLH24-82-L

(searched

only in

human

protein

dataset)

Match to: Q53H99_HUMAN Score: 17248 Inhibitor of DNA binding 2 variant (Fragment).- Homo sapiens (Human)

Found in search of C:\Documents and Settings\Administrator\Desktop\Marie\1D2_BHLH.RAW Nominal mass (M r): 15071; Calculated pI value: 7.82

NCBI BLAST search of Q53H99_HUMAN against nr Unformatted sequence string for pasting into other applications Taxonomy: Homo sapiens Links to retrieve other entries containing this sequence from NCBI Entrez: BAD96402 from Homo sapiens Fixed modifications: Carbamidomethyl (C)

Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P

Sequence Coverage: 25%

Matched peptides shown in Bold Red

1 MKAFSPVRSV RKYSLSDHSL GISR SKTPVD DPMSLLYNMN DCYSKLKELV

101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG

3.4 Crystallization

Crystal screens were done initially in a high-throughput 96-well sitting-drop format using an Innovadyne robot to dispense the screening solutions and protein at 200 nl volumes This enabled many different screens from both Qiagen and Hampton

Research to be tested HLH24-82-L was screened and the best hit was found with the Cation Suite from Qiagen In the condition 0.1 M MES pH 6.5, 2.5 M Lithium Acetate, crystals were small, needle like and tended to clump together To optimize growth, manual hanging-drop screens were setup in a larger final volume of 2 µl to allow more space for growth At 18°C, the crystals were still quite small and clumpy (Figure 10A) There was no significant difference in the size of crystals grown at different temperatures but those at 18°C had the sharpest edges so this temperature was maintained for further optimization steps To try obtaining a single crystal that had a large enough needle that could be broken off for mounting, microseeding technique was used to aid in nucleation The crystals in Figure 10A were crushed and serial dilutions made from the stock solution were then used to setup the drops

of 1 µl of the diluted mixture with 1 µl of precipitant solution and 1 µl protein solution With increasing dilutions, there were fewer crystals, but they were larger and had fewer needles (Figure 10, B-D) Single needles were carefully broken off with a cryoloop and flash cooled in liquid nitrogen for X-ray diffraction studies