Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation.

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.

Specific and Selective Inhibitors of Proprotein Convertases Engineered by Transferring Serpin B8 Reactive-Site and Exosite Determinants of Reactivity to the Serpin α1PDX.

The molecular determinants of substrate specificity and selectivity in the proprotein convertase (PC) family of proteases are poorly understood. Here we demonstrate that the natural serpin family inhibitor, serpin B8, is a specific and selective inhibitor of furin relative to the other PCs of the constitutive protein secretion pathway, PC4, PC5, PACE4, and PC7 (PC4-PC7, respectively), and identify reactive-site (P6-P5' residues) and exosite elements of the serpin that contribute to this specificity and selectivity through studies of chimeras of serpin B8 and α1PDX, an engineered serpin inhibitor of furin. Kinetic studies revealed that the specificity and selectivity of the serpin chimeras for inhibiting PCs were determined by P6-P5 and P3-P2 residue-dependent recognition of the P4Arg-X-X-P1Arg PC consensus sequence and exosite-dependent recognition of the reactive loop P2' residue of the chimeras by the PCs. Both productive and nonproductive binding of the chimeras to PC4-PC7 but not to furin contributed to a decreased specificity for inhibiting PC4-PC7 and an increased selectivity for inhibiting furin. Molecular dynamics simulations suggested that nonproductive binding of the chimeras to the PCs was correlated with a greater conformational variability of the catalytic sites of PC4-PC7 relative to that of furin. Our findings suggest a new approach for designing selective inhibitors of PCs using α1PDX as a scaffold, as evidenced by our ability to engineer highly specific and selective inhibitors of furin and PC4-PC7.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability.

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their ß-turn occurrence, we engineered two chimerical enzymes where their super secondary ß-loop-α motifs 2 ((ßα)2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (ßα)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (ßα)2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (ßα)2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA ). A systematic analysis of DSC data showed a large decrease on the EA of TcTIM (ΔEA ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.

Interplay between Protein Thermal Flexibility and Kinetic Stability.

Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (ß/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.