The influence of ANS on the dynamic light scattering of 1 µM DPGN in the presence of 10 mM (pH adjusted) TCEP.

<p>The solid squares are in the presence of 20 µM ANS whereas the solid circles are in its absence.</p

Reduction of 1.8 µM DPGN leads to precipitation.

<p>(Top panel) Plasminogen was treated with mercaptoethanol, dithiothreitol or TCEP at the concentrations shown in the figure. Precipitation was followed by monitoring the change in light scattering/Absorbance at 280 nm. The starting absorbance was about 0.4. Each curve has been offset for ease of viewing. The buffer was 5 mM K₂HPO₄, 5 mM KH₂PO₄, 100 mM NaCl, pH 7; the temperature was maintained at 20°C. (Bottom panel) At the times indicated in the figure, 0.1 mL of the sample containing 0.133 M dithiothreitol was removed from the cuvet and immediately centrifuged at 13 000 rpm. The precipitate and supernate were separated. SDS sample buffer was added to each. Both were subjected to SDS-PAGE on 7% gels. The precipitates are shown in the figure. The gels of the supernates were not sufficiently stained with Coomassie Brilliant Blue as to make the plasminogen easily visible.</p

Precipitation of plasminogen by DTT/Dihydroxydithiane anaerobic mixtures.

<p>From left to right, the ratios are 1000∶1, 100∶1, 10∶1, 1∶1, 1∶10, 1∶100, 1∶1000. At 1∶10, about 50% of the plasminogen precipitates. E' (as an oxidation potential) of the mixture at this ratio is about 0.3 V. This gives a driving force of about 14 kcal for reduction of the plasminogen.</p

6-aminohexanoic acid inhibits the precipitation reaction.

<p>DPGN (2.3 µM) was treated with 15 mM dithiothreitol in the presence or absence of 26 mM 6-aminohexanoate. The buffer conditions were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006196#pone-0006196-g001" target="_blank">Figure 1</a>.</p

The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus

<div><p>The enolase produced by <i>Streptococcus pyogenes</i> is a homo-octamer whose overall shape resembles that of a donut. The octamer is best described as a tetramer of dimers. As such, it contains two types of interfaces. The first is common to almost all enolases as most enolases that have been studied are dimers. The second is unique to the octamers and includes residues near the carboxy-terminus. The primary sequence of the enolase contains 435 residues with an added 19 as an N-terminal hexahistine tag. We have systematically truncated the carboxy-terminus, individually removing the first 8 residues. This gave rise to a series of eight structures containing respectively, 435, 434, 433, 432, 431, 430, 429 and 427 residues. The truncations cause the protein to gradually dissociate from octamers to enzymatically inactive monomers with very small amounts of intermediate tetramers and dimers. We have evaluated the contributions of the missing residues to the monomer/octamer equilibrium using a combination of analytical ultracentrifugation and activity assays. For the dissociation reaction,</p><p></p><p></p><p></p><p><mi>o</mi><mi>c</mi><mi>t</mi><mi>a</mi><mi>m</mi><mi>e</mi><mi>r</mi><mo> </mo></p><p><mo>⇐</mo><mrow></mrow></p><p><mo>⇒</mo><mrow></mrow></p><mo> </mo><mn>8</mn><mo> </mo><mi>m</mi><mi>o</mi><mi>n</mi><mi>o</mi><mi>m</mi><mi>e</mi><mi>r</mi><p></p><p></p><p></p><p></p>truncation of all eight C-terminal residues resulted in a diminution in the standard Gibbs energy of dissociation of about 59 kJ/mole of octamer relative to the full length protein. Considering that this change is spread over eight subunits, this translates to a change in standard Gibbs interaction energy of less than 8 kJ/mole of monomer distributed over the eight monomers. The resulting proteins, containing 434, 433, 432, 431, 430, 429 and 427 residues per monomer, showed intermediate free energies of dissociation. Finally, three other mutations were introduced into our reference protein to establish how they influenced the equilibrium. The main importance of this work is it shows that for homo-multimeric proteins a small change in the standard Gibbs interaction energy between subunits can have major physiological effects.<p></p></div

Str enolase (3ZLH.PDB) showing the alternating pattern of interfaces.

<p>The red represents residues 428–433 of the protein and indicates the position of the dimer-dimer interface; residues 434–435 do not appear in the X-ray structure and are not shown. The other interface is that of the monomer-monomer. Left side panel. Residues 428–433 appear on four subunits looking down on the donut and on the alternating subunits on the other side of the donut. Right side panel. Looking from the side of the donut clearly shows the nature of the dimer-dimer interface.</p

The hole which would be created but is here covered by the eight (red) c-terminal amino acid residues from Str enolase 137/363.

<p>Of the visible residues removed, three are polar, one is hydrophobic and one is mixed. The two terminal lysines are not visible but are clearly polar.</p

The truncation of Str enolase 137/363 from the carboxy-terminus results in dissociation of the octamer and the concomitant formation of monomers.

<p>The reference sample (black curve) was Str enolase 137/363. All samples were prepared in TME-SO₄. In the truncated samples, the data at s > 18 are not shown. These are aggregates and do not contribute to the calculation of either Kd or ΔG°. Since there are more aggregates in the -4, -6 and -8 truncations than in the -2, the monomer peak in the -2 sample is larger than in the other truncations. The samples chosen for the five curves were chosen because they corresponded to the average values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135754#pone.0135754.t001" target="_blank">Table 1</a>.</p

Thermodynamic parameters of the mutated and truncated forms of Str enolase.

<p>Thermodynamic parameters of the mutated and truncated forms of Str enolase.</p

Activity and CD characteristics of mutated and truncated forms of Str enolase.

<p>ND = not determined,</p><p>* The standard errors as a percentage of the activity for these samples are high because the concentration of protein in the assays is very high and there is reactivation of the enolase during the assay.</p><p>Activity and CD characteristics of mutated and truncated forms of Str enolase.</p