Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal Proteins L1.

In our previous papers, we proposed the idea that programs predicting intrinsically disordered regions in amino acid sequences can be used for finding weakened sites in proteins. The regions predicted by such programs are suitable targets for the introduction of protein-stabilizing mutations. However, for each specific protein, it remains unclear what determines protein stabilization - the amino acid sequence (and accordingly, prediction of weakened sites) or the 3D structure. To answer this question, it is necessary to study two proteins with similar structures but different amino acid sequences and, consequently, different predictions of weakened regions. By introducing identical mutations into identical elements of the two proteins, we will be able to reveal whether predictions of the weakened sites or the 3D protein structure are the key factors in the protein stability increase. Here, we have chosen ribosomal proteins L1 from the halophilic archaeon Haloarcula marismortui (HmaL1) and extremophilic bacterium Aquifex aeolicus (AaeL1). These proteins are identical in their structure but different in amino acid sequences. A disulfide bond introduced into the region predicted as the structured one in AaeL1 did not lead to the increase in the protein melting temperature. At the same time, a disulfide bond introduced into the same region in HmaL1 that was predicted as a weakened one, resulted in the increase in the protein melting temperature by approximately 10°C.

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.