Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide- and protein-binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non-proteinaceous ligand-binding protein, flavin-based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids so that the resulting sequences are 55%-66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.

Small-angle X-ray scattering structural insights into alternative pathway of actin oligomerization associated with inactivated state.

In addition to the well-known monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can exist in the so-called inactivated form (I-actin). Hsp70 chaperon, prefoldin, and CCT chaperonin are required to obtain native globular state. In contrast, I-actin is spontaneously formed in the absence of intracellular folding machinery. I-actin can be obtained from G-actin by elimination of divalent ion, incubation in presence of small concentrations of denaturants, and by heat exposure. Since G-actin is a quasi-stationary, thermodynamically unstable form, it can gradually transform into inactivated state in the absence of chelating/denaturating agents or heat exposure, but the transition is much slower. I-actin was shown to associate into oligomers up to the molecular weight of 14-16 G-actin monomers, though the structure of these oligomers remains uncharacterized. This study employs small-angle X-ray scattering to reveal novel insights into the oligomerization process of such spontaneously formed inactivated actin. These oligomers are differentiated from F-actin through comparative analysis, highlighting a unique oligomerization pathway.