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

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(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.
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37!
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)

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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)

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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
1 MKAFSPVRSV RKNSLSDHSL GISRSKTPVD DPMSLLYNMN DCYSKLKELV
51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR
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 GISRSKTPVD DPMSLLYNMN DCYSKLKELV
51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR
101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG
HLH24-82-L-
Se-Met

(searched
against all
non-
redundant
proteins with
Se variable
modification
set)
Match to: IPI00294210 Score: 1435

Tax_Id=9606 Gene_Symbol=ID2 DNA-binding protein inhibitor ID-2
Found in search of C:\Documents and
Settings\Administrator\Desktop\Se_Met_C3_1.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),Delta:S (-1)Se (1) (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 RKNSLSDHSL GISRSKTPVD DPMSLLYNMN DCYSKLKELV
51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR
101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG
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40!
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.