5HKG

Total chemical synthesis, refolding and crystallographic structure of a fully active immunophilin: calstabin 2 (FKBP12.6).

Summary for 5HKG

• Entry DOI: 10.2210/pdb5hkg/pdb
• Related: 1C9H
• Descriptor: Peptidyl-prolyl cis-trans isomerase FKBP1B, RAPAMYCIN IMMUNOSUPPRESSANT DRUG, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: synthetic protein, refolding, immunophilin, isomerase
• Biological source: Homo sapiens (Human)
• Cellular location: Cytoplasm : P68106
• Total number of polymer chains: 1
• Total formula weight: 12936.11

Authors

Sirigu, S.,Huet, T.,Bacchi, M.,Jullian, M.,Fould, B.,Ferry, G.,Vuillard, L.,Chavas, L.,Puget, K.,Nosjean, O.,Boutin, J.A. (deposition date: 2016-01-14, release date: 2016-10-05, Last modification date: 2024-01-10)

Primary citation

Bacchi, M.,Jullian, M.,Sirigu, S.,Fould, B.,Huet, T.,Bruyand, L.,Antoine, M.,Vuillard, L.,Ronga, L.,Chavas, L.M.,Nosjean, O.,Ferry, G.,Puget, K.,Boutin, J.A.
Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6).
Protein Sci., 25:2225-2242, 2016

PubMed Abstract: Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

PubMed: 27670942
DOI: 10.1002/pro.3051

Experimental method

X-RAY DIFFRACTION (1.5 Å)