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The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2).

Frost L, Baez MA, Harrilal C, Garabedian A, Fernandez-Lima F, Leng F - PLoS ONE (2015)

Bottom Line: It consists of three positively charged "AT-hooks" and a negatively charged C-terminus.Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure.Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States of America; Department of Chemistry and Biochemistry, Florida International University, Miami, Florida, United States of America.

ABSTRACT
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged "AT-hooks" and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

No MeSH data available.


Related in: MedlinePlus

(A) A typical 2D IMS-MS contour plot showing the monomer and dimer signals at 100 μM.Notice the overlap at even charge states in the MS domain for the monomer and dimer peaks. (B) Relative abundance of the dimer formation as a function of the concentration for the odd charge states.
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pone.0130478.g004: (A) A typical 2D IMS-MS contour plot showing the monomer and dimer signals at 100 μM.Notice the overlap at even charge states in the MS domain for the monomer and dimer peaks. (B) Relative abundance of the dimer formation as a function of the concentration for the odd charge states.

Mentions: The analysis of 2D IMS-MS contour plots (Fig 4) showed the separation in mobility and m/z domains of the [M+nH]+n and [2M+nH]+n monomer and dimer charge state distributions, respectively. Close inspection of the 2D IMS-MS plots shows that the for odd charge state of the [2M+nH]+n series, a clear separation of the dimer component can be achieved, since there is no equivalent m/z on the [M+nH]+n monomer series. In addition, the IMS-MS plots permit the measurements of the relative abundance of each series without the interferences from other m/z. Analysis of the relative abundances of the dimer series relative to the monomer series as a function of the protein concentration in the ESI starting solution shows that as the concentration increases, the dimer formation increases. In particular, dimer observation is significant at concentrations larger than 10 μM. While the mechanism of dimer formation maybe be controversial (i.e., pre-formed in solution or formed during ESI process in the gas-phase), our experimental data from previous experiments (see above) suggest that dimers are pre-formed in solution and remain intact during the ESI process (soft ionization). The lack of trimer, tetramer and higher order oligomers typically observed during ESI condensation at high sample concentration in the IMS-MS data also supports this hypothesis.


The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2).

Frost L, Baez MA, Harrilal C, Garabedian A, Fernandez-Lima F, Leng F - PLoS ONE (2015)

(A) A typical 2D IMS-MS contour plot showing the monomer and dimer signals at 100 μM.Notice the overlap at even charge states in the MS domain for the monomer and dimer peaks. (B) Relative abundance of the dimer formation as a function of the concentration for the odd charge states.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482583&req=5

pone.0130478.g004: (A) A typical 2D IMS-MS contour plot showing the monomer and dimer signals at 100 μM.Notice the overlap at even charge states in the MS domain for the monomer and dimer peaks. (B) Relative abundance of the dimer formation as a function of the concentration for the odd charge states.
Mentions: The analysis of 2D IMS-MS contour plots (Fig 4) showed the separation in mobility and m/z domains of the [M+nH]+n and [2M+nH]+n monomer and dimer charge state distributions, respectively. Close inspection of the 2D IMS-MS plots shows that the for odd charge state of the [2M+nH]+n series, a clear separation of the dimer component can be achieved, since there is no equivalent m/z on the [M+nH]+n monomer series. In addition, the IMS-MS plots permit the measurements of the relative abundance of each series without the interferences from other m/z. Analysis of the relative abundances of the dimer series relative to the monomer series as a function of the protein concentration in the ESI starting solution shows that as the concentration increases, the dimer formation increases. In particular, dimer observation is significant at concentrations larger than 10 μM. While the mechanism of dimer formation maybe be controversial (i.e., pre-formed in solution or formed during ESI process in the gas-phase), our experimental data from previous experiments (see above) suggest that dimers are pre-formed in solution and remain intact during the ESI process (soft ionization). The lack of trimer, tetramer and higher order oligomers typically observed during ESI condensation at high sample concentration in the IMS-MS data also supports this hypothesis.

Bottom Line: It consists of three positively charged "AT-hooks" and a negatively charged C-terminus.Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure.Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States of America; Department of Chemistry and Biochemistry, Florida International University, Miami, Florida, United States of America.

ABSTRACT
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged "AT-hooks" and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

No MeSH data available.


Related in: MedlinePlus