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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

Self-association of HMGA2 was demonstrated by the FRET experiments.(A) and (C), respectively, represent fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR in 50 mM Tris-HCl (pH 8.0) and 50 mM (A) or 200 mM (C) NaCl. (B) and (D) are the difference in fluorescence intensity at 518 nm (ΔF) as a function of HMG-TMR concentration was shown for the FRET experiment in panel A and panel C respectively. The fluorescence spectra of HMGA2-FM (20 nM; λexcitation = 492 nm) were recorded as described under “Materials & Methods.”
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pone.0130478.g003: Self-association of HMGA2 was demonstrated by the FRET experiments.(A) and (C), respectively, represent fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR in 50 mM Tris-HCl (pH 8.0) and 50 mM (A) or 200 mM (C) NaCl. (B) and (D) are the difference in fluorescence intensity at 518 nm (ΔF) as a function of HMG-TMR concentration was shown for the FRET experiment in panel A and panel C respectively. The fluorescence spectra of HMGA2-FM (20 nM; λexcitation = 492 nm) were recorded as described under “Materials & Methods.”

Mentions: The self-association of HMGA2 was further studied by fluorescence resonance energy transfer (FRET) titration experiment. In this experiment, HMGA2 was labeled with fluorescein-5-maleimide (FM) or tetramethylrhodamin-5-maleimide (TMR) to produce HMGA2-FM or HMGA2-TMR. If HMGA2 self-associates into homodimers or homooligomers, FRET should be detected using the FM donor/TMR acceptor fluorophore pair. Fig 3 shows the fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR for two different salt concentrations, 50 mM (Fig 3A and 3B) and 200 mM NaCl (Fig 3C and 3D). The fluorescence intensity at 518 nm decreased with increasing HMGA2-TMR concentrations. The decrease saturated at high HMGA2-TMR concentration, indicating that HMGA2-TMR binds to HMGA2-FM. Interestingly, we observed some difference between the two FRET experiments. For the FRET experiment in the presence of 50 mM NaCl, the fluorescence decrease saturated at 5 to 10 nM of HMGA2-TMR and increased again after adding more than 20 nM of HMGA2-TMR (Fig 3B). For the FRET experiment in the presence of 200 mM NaCl, the fluorescence decrease saturated at 20 nM of HMGA2-TMR, indicating a 1:1 molar ratio of HMGA2-FM/HMGA2-TMR (Fig 3B). We also observed the fluorescence increase at 588 nm and an isobestic point for the experiment in the presence of 200 mM NaCl (Fig 3C). In addition, we found that the quench magnitude is different. For the 50 mM case, more than 50% of HMGA2-FM fluorescence was quenched by HMGA2-TMR (Fig 3A); for 200 mM case, only about 10% of HMGA2-FM fluorescence was quenched by HMGA2-TMR (Fig 3C). These results suggest that the self-association of HMGA2 is salt-dependent and the electrostatic interaction plays an important role in the self-association process.


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)

Self-association of HMGA2 was demonstrated by the FRET experiments.(A) and (C), respectively, represent fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR in 50 mM Tris-HCl (pH 8.0) and 50 mM (A) or 200 mM (C) NaCl. (B) and (D) are the difference in fluorescence intensity at 518 nm (ΔF) as a function of HMG-TMR concentration was shown for the FRET experiment in panel A and panel C respectively. The fluorescence spectra of HMGA2-FM (20 nM; λexcitation = 492 nm) were recorded as described under “Materials & Methods.”
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130478.g003: Self-association of HMGA2 was demonstrated by the FRET experiments.(A) and (C), respectively, represent fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR in 50 mM Tris-HCl (pH 8.0) and 50 mM (A) or 200 mM (C) NaCl. (B) and (D) are the difference in fluorescence intensity at 518 nm (ΔF) as a function of HMG-TMR concentration was shown for the FRET experiment in panel A and panel C respectively. The fluorescence spectra of HMGA2-FM (20 nM; λexcitation = 492 nm) were recorded as described under “Materials & Methods.”
Mentions: The self-association of HMGA2 was further studied by fluorescence resonance energy transfer (FRET) titration experiment. In this experiment, HMGA2 was labeled with fluorescein-5-maleimide (FM) or tetramethylrhodamin-5-maleimide (TMR) to produce HMGA2-FM or HMGA2-TMR. If HMGA2 self-associates into homodimers or homooligomers, FRET should be detected using the FM donor/TMR acceptor fluorophore pair. Fig 3 shows the fluorescence spectra of HMGA2-FM in the presence of increasing concentrations of HMGA2-TMR for two different salt concentrations, 50 mM (Fig 3A and 3B) and 200 mM NaCl (Fig 3C and 3D). The fluorescence intensity at 518 nm decreased with increasing HMGA2-TMR concentrations. The decrease saturated at high HMGA2-TMR concentration, indicating that HMGA2-TMR binds to HMGA2-FM. Interestingly, we observed some difference between the two FRET experiments. For the FRET experiment in the presence of 50 mM NaCl, the fluorescence decrease saturated at 5 to 10 nM of HMGA2-TMR and increased again after adding more than 20 nM of HMGA2-TMR (Fig 3B). For the FRET experiment in the presence of 200 mM NaCl, the fluorescence decrease saturated at 20 nM of HMGA2-TMR, indicating a 1:1 molar ratio of HMGA2-FM/HMGA2-TMR (Fig 3B). We also observed the fluorescence increase at 588 nm and an isobestic point for the experiment in the presence of 200 mM NaCl (Fig 3C). In addition, we found that the quench magnitude is different. For the 50 mM case, more than 50% of HMGA2-FM fluorescence was quenched by HMGA2-TMR (Fig 3A); for 200 mM case, only about 10% of HMGA2-FM fluorescence was quenched by HMGA2-TMR (Fig 3C). These results suggest that the self-association of HMGA2 is salt-dependent and the electrostatic interaction plays an important role in the self-association process.

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