9RXF
E20K/N28G/V36L/D43K/Q48E/I59A/E61K/E72K/V76L/N79S/I92A/D126K/A142V/D153K/D154E/S158T FLAVODOXIN FROM ANABAENA
Summary for 9RXF
| Entry DOI | 10.2210/pdb9rxf/pdb |
| Descriptor | Flavodoxin, FLAVIN MONONUCLEOTIDE, GLYCEROL, ... (4 entities in total) |
| Functional Keywords | electron transport |
| Biological source | Nostoc sp. PCC 7119 |
| Total number of polymer chains | 2 |
| Total formula weight | 38618.60 |
| Authors | Martinez-Julvez, M.,Perez-Correa, V.,Sancho, J.,Hidalgo-Toledo, A. (deposition date: 2025-07-11, release date: 2025-11-05) |
| Primary citation | Hidalgo-Toledo, A.,Bazco, D.,Correa-Perez, V.,Martinez-Julvez, M.,Sancho, J. Protein thermostabilization with Protposer: Pushing the stability limits and folding reversibility of a highly-stabilized apoflavodoxin. Int.J.Biol.Macromol., 331:148333-148333, 2025 Cited by PubMed Abstract: Enhancing the stability of highly stable proteins represents an interesting challenge in protein design. We have used the computational tool Protposer to rapidly achieve large additional stabilization of apoflavodoxin, a protein strongly thermostabilized over the years through protein engineering based on educated guesses. By rationally combining top-ranked mutations onto a previously stabilized variant (6 M), we have generated a series of new mutants and characterized their stability by thermal and chemical denaturation. Relative to the starting variant, the T of 10 M apoflavodoxin is nearly 9 °C higher, while the simplified 3 M and 4 M mutants, showing improved refolding properties, display increases of 6/7.5 °C, respectively. The thermostabilizing effects of individual mutations are close to additive and translate into a large increase in conformational stability at 25.0 °C. Comparison of the x-ray structures of progressively stabilized WT, 6 M and 10 M flavodoxins reveals a concomitant mild trend toward shorter hydrogen bonds, reduced internal cavity volumes and narrower tunnels. Overall, these conformational changes are minor, and a functional assay confirms the mutants also preserve their catalytic activity. These findings demonstrate that even highly stable proteins can be further stabilized through rational design using a simple computational tool that automatically analyses PDB files and identifies stabilizing mutations. PubMed: 41106744DOI: 10.1016/j.ijbiomac.2025.148333 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.4 Å) |
Structure validation
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