Arrhenius plots of GFP<i>-</i>cycle3 and its mutant forms.

<p>Dependencies of logarithms of rate constants of the first (<i>k</i>₁) (solid line in the figures) and second (<i>k</i>₂) (dashed line) stages of GFP<i>-</i>cycle3 heat denaturation versus inverse temperature calculated from melting curves of GFP<i>-</i>cycle3 (gray lines) and its mutant forms (colored lines). <b>A</b>, the plots are related to singular substitutions of hydrophobic amino acids. <b>B</b>, the plots are related to double substitutions for cysteines with an ss-bridge between them. <b>C</b>, the plots are related to double substitutions for cysteines modified with iodoacetamide (an ss-bridge is not formed).</p

GFP<i>-</i>cycle3 structure formation.

<p>Sequence of formation/distortion of secondary structure elements of GFP<i>-</i>cycle3 based on the results of multimutational analysis. <i>N, I₁, I₂, D</i>, and <i>U</i> are native, two intermediate, denatured and unfolded states of the protein, respectively. At the first stage of unfolding (<i>N</i>→<i>I₁</i>) the packing of amino acids on the protein surface is impaired, but the packing of the hydrophobic nucleus as well as the entire packing of the protein chain are not distorted. During the following stage (I<i>₁</i>→<i>I₂</i>) the structure of β-strands (shown in yellow color) is changed (in the region of amino acids 90–130). After that the protein unfolds almost completely (<i>I₂</i>→<i>D</i>), excluding three β-strands in the region of amino acids 10–50 which at “mild” denaturation remain structured in the unfolding state. The last stage (<i>D</i>→<i>U</i>), when the total protein acquires a coil-like conformation, is probable only under the action of strong denaturants.</p

Rate constants of GFP-cycle3 unfolding obtained with the fluorescence method is well compatible with the same obtained with the calorimetry method.

<p>(<b>A</b>) Typical kinetic unfolding curves of GFP-cycle3 at pH 6.2 caused by a temperature jump from 293.2 K to 346.5 K, 349.2 K and 352.6 K, accordingly. The curves (noisy lines) were well fitted to two exponentials (continuous lines). Residual plot for fitting is shown in the upper panel. (<b>B</b>) Dependences of the logarithm of rate constants (<i>k₁</i> and <i>k₂</i>) of unfolding of GFP-cycle3 versus reverse temperature. Rate constants obtained with the fluorescence method are shown by symbols. Rate constants obtained from calorimetric experiments are shown by lines.</p

What changes in energy levels of a multi-state protein could be expected if we make single mutation or ss-bridge incorporation.

<p>Schematic representation of the sequence of states at unfolding of GFP (on the left) and relative arrangement of energetic levels (on the right) of this protein. <i>N</i>, <i>I</i>, <i>D</i>, and <i>U</i> are native, intermediate, denatured and unfolded states of the protein, respectively. Substituted amino acid residues (mutations) are shown with black circles. <b>S1</b>, the substituted amino acid residue is contained only in the structure of native state. Therefore destabilization of the native state should lead to acceleration of only the first stage of unfolding (<i>k</i>1). <b>S2</b>, the substituted amino acid residue is included in the structure of the very last state (<i>D</i>) hence it should affect the stability of all states and also the rates of all unfolding stages. <b>S3</b>, the ss-bridge (two black circles in the figure) in the structured part of state <i>D</i> would influence only the very last stage of unfolding because it does not affect the mobility of states <i>N</i> and <i>I</i>. It is assumed that we have introduced an ideal ss-bridge that has not impaired the protein internal packing. <b>S4</b>, the ss-bridge in the structured part of state <i>N</i> would influence all the stages of unfolding because it “forces” the protein region that should unfold during the first stage to be compact. There is an intentional inaccuracy in the represented energetic schemes. In fact, if an intermediate state <i>I</i> (for example in S2) is destabilized by a mutation, the free energy of the transition state between <i>N</i> and <i>I</i> should change at least by the same value. In this figure, the energetic schemes are represented in assumption that every unfolding stage occurs independently of all the other processes. Since only the unfolding rates are analyzed here, such a simplification of the energetic schemes is quite reasonable and provides an easy way to compare these rates with each other and to relate theoretical considerations with the experimental data.</p

Choice of mutations in GFP-cycle3.

<p>End view (A) and side view (B) of the voluminous model of the GFP-can. Four structure elements are shown individually: (C–E) three β-sheets and (F) β-hairpin. Balls denote amino acids substituted for alanine (I14, V112, I161, L201) and pairs of amino acids substituted for cysteines (V11 and D36, V93 and Q111, K162 and Q184, S202 and T225). Plot G shows the amount of residue-residue contacts of each amino acid residue (a contact distance is 6 Å. Black columns denote hydrophobic amino acids. Coloured lines at the bottom of plot G denote the position of β-strands in structure elements C–F. Coloured circles in plot G show columns referring to amino acids substituted for alanine. Coloured circles connected by a line at the bottom of the plot mark pairs of amino acids substituted for cysteines.</p

Effect of single amino acids substitutions on GFP<i>-</i>cycle3 unfolding rate constants.

<p>Change of logarithms of unfolding rate constants for GFP<i>-</i>cycle3 mutant proteins with single amino acids substitutions as compared to the WT (GFP<i>-</i>cycle3) protein: ΔLn(<i>k</i>_1,2) = ΔLn(<i>k</i>_1,2)_mut− ΔLn(<i>k</i>_1,2)_WT.</p

Combined fitting parameters of heat denaturation curves of GFP<i>-</i>cycle3 and its mutants using a model involving two consecutive irreversible steps of denaturation (eq. 9, 10).

<p>Fitting errors are given. See experimental errors in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048604#s4" target="_blank">Materials and methods</a>.</p

GFP-cycle3 melting and choice of irreversible denaturation models.

<p>Dependences of excess molar heat capacity versus temperature for GFP-cycle3 measured at three scanning rates (1.0, 0.5, 0.25 K/min). Symbols show experimental data and lines show data calculated with the use of one-stage model (A), the Lumry-Eyring model with the fast equilibrating first step (B) and irreversible models involving two consecutive irreversible steps of denaturation (C). Residual plot for fitting is shown in the upper panels.</p