(A) Amino acid sequence of mouse HMGA2.

<p>The positively charged “AT hooks” and the negatively charged C-terminus are highlighted by underlines and a box, respectively. <b>(B) The experimental (open dots with dotted line) and calculated (solid line) CD spectra of HMGA2 in BPES buffer.</b> The CD spectra were calculated by the program CONTIN as described in the text. The solid dots are residuals. <b>(C) Gel filtration chromatography profile of HMGA2 in solution.</b> Recombinant HMGA2 was resolved by gel filtration in the Sephacryl S-100 column as described under “Materials and Methods.” V₀ is the void volume; A, C, and R represent the elution volume of albumin, chymotrypsinogen A, and ribonuclease A, respectively.</p

Chemical cross-linking of HMGA2 and HMGA2Δ95–108 with EDC.

<p>Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained HMGA2Δ95–108 in the absence of EDC; lanes 2 and 3 contained 40 μM HMGA2Δ95–108 in the presence of 2 mM EDC; lane 4 contained HMGA2 in the absence of EDC; lanes 5 and 6 contained 40 μM HMGA2 in the presence of 2 mM EDC; lane 7 contained molecular standards.</p

Self-association of HMGA2 was demonstrated by the FRET experiments.

<p><b>(A)</b> and <b>(C)</b>, 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 <b>(A)</b> or 200 mM <b>(C)</b> NaCl. <b>(B)</b> and <b>(D)</b> 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 <b>A</b> and panel <b>C</b> respectively. The fluorescence spectra of HMGA2-FM (20 nM; λ_excitation = 492 nm) were recorded as described under “Materials & Methods.”</p

Hydrodynamic properties of HMGA2 calculated from sedimentation velocity results by program Sednterp.

<p>^aThe apparent molecular weight (Mw) and Stokes’ radius (R_Stokes) of HMGA2 was calculated according to sedimentation velocity studies.</p><p>^bThe apparent Mw and R_Stokes of HMGA2 were calculated according to gel-filtration results. In this table, we assume that HMGA2 is a cylinder. Two parameters, L, the length of the cylinder and d, the diameter, are required to calculate the hydrodynamic model. The following three equations are used to compute L and d: </p><p></p><p></p><p><mi>s</mi><mo>=</mo></p><p></p><p><mi>M</mi><mo stretchy="false">(</mo><mn>1</mn><mo>−</mo></p><p><mi>ν</mi><mo>¯</mo></p><mi>ρ</mi><mo stretchy="false">)</mo><p></p><p></p><p><mi>N</mi><mn>0</mn></p><mi>f</mi><p></p><p></p><p></p><p></p><p></p>; <p></p><p></p><p><mi>f</mi><mo>=</mo></p><p></p><p><mn>3</mn><mi>π</mi><mi>η</mi><mi>L</mi></p><p><mi>ln</mi></p><p><mo>{</mo></p><p></p><p><mi>L</mi><mi>d</mi></p><mo>+</mo><p><mo>[</mo></p><p><mn>0.312</mn><mo>+</mo></p><p></p><p><mn>0.561</mn><mi>d</mi></p><mi>L</mi><p></p><mo>+</mo><mn>0.1</mn><p></p><p><mo stretchy="false">(</mo></p><p><mi>d</mi><mi>L</mi></p><mo stretchy="false">)</mo><p></p><mn>2</mn><p></p><p></p><mo>]</mo><p></p><p></p><mo>}</mo><p></p><p></p><p></p><p></p><p></p><p></p>; <p></p><p></p><p><mi>V</mi><mo>=</mo></p><p></p><p><mi>M</mi></p><p><mi>δ</mi><mn>1</mn></p><p></p><p></p><p><mi>N</mi><mn>0</mn></p><p><mi>ρ</mi><mrow></mrow></p><p></p><p></p><mo>+</mo><p></p><p><mi>M</mi></p><p><mi>ν</mi><mo>¯</mo></p><p></p><p></p><p><mi>N</mi><mn>0</mn></p><p></p><p></p><mo>=</mo><mi>π</mi><p></p><p><mo stretchy="false">(</mo></p><p><mi>d</mi><mn>2</mn></p><mo stretchy="false">)</mo><p></p><mn>2</mn><p></p><mi>L</mi><p></p><p></p><p></p>, where M, s, f, v-bar, and N₀ are the molecular weight of the solute (protein), the sedimentation coefficient, the friction coefficient, the partials specific volume of the protein, the buffer density, the buffer viscosity, and Avogadro’s number, respectively.<p></p><p>Hydrodynamic properties of HMGA2 calculated from sedimentation velocity results by program Sednterp.</p

Chemical cross-linking HMGA2 into homodimers with EDC.

<p>Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained molecular standards; lane 2 contained HMGA2 in the absence of EDC; lanes 3 to 6 contained, respectively, 29, 39, 58, and 116 μM HMGA2 in the presence of 2 mM EDC. M, monomer; D, dimer; T3, trimer; T4, tetramer.</p

The CTP-TMR and HMGA2Δ95–108 co-eluting in gel-filtration chromatography.

<p>The CTP-TMR was prepared as described under “Materials and Methods” and incubated with HMGA2Δ95–108 at 24°C for 30 min in BPES buffer. The CTP-TMR and HMGA2Δ95–108 mixture was then subjected to a Sephacryl S-100 HR filtration column (1×50 cm) equilibrated with BPES buffer. Gel filtration profile of the CTP-TMR binding to HMGA2Δ95–108 was monitored by a graph of OD₅₅₆ versus elution volume (A) and a 15% SDS PAGE gel (B). Lanes 1 to 8 of the SDS-PAGE gel (B) correspond to the fractions 1 to 8 labeled in panel a. Free HMGA2Δ95–108 and the CTP-TMR were eluted at 22 and 30 ml respectively in the column.</p

(A) A typical 2D IMS-MS contour plot showing the monomer and dimer signals at 100 μM.

<p>Notice the overlap at even charge states in the MS domain for the monomer and dimer peaks. <b>(B) Relative abundance of the dimer formation as a function of the concentration for the odd charge states.</b></p

Results for individually fitting the sedimentation velocity data to the model of a single ideal species by the program Sedfit (version 8.7).

<p>^aIn this table, <i>s</i> represents the sedimentation coefficient of HMGA2 at 20°C; Mw represents molecular weight; RMSD of fit is the root-mean-square deviation of the fit.</p><p>^bValues in parentheses are the 95% confidence interval (CI) for the molecular weight and sedimentation coefficient.</p><p>Results for individually fitting the sedimentation velocity data to the model of a single ideal species by the program Sedfit (version 8.7).</p

A possible model for the HMGA2 homodimerization.

<p>Blue lines represent the protein backbone. Electrostatic interactions between the positively charged “AT hooks” (red rectangle with two red circles) and the negatively charged C-terminus (yellow oval) coordinate the dimer formation. <b>(A)</b> represents HMGA2 monomers. <b>(B)</b> and <b>(C)</b> represent different interchangeable conformations of HMGA2 homodimers. <b>(C)</b> is more consistent with our EDC cross-linking and sedimentation velocity results. The HMGA2 homodimers may be an ensemble of different conformers.</p