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

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 structurefunction relationship of enzymes reachable by complete chemical synthesis.

General lack of structural characterization of chemically synthesized long peptides

“…The refolding process, used on the basis of several years of refolding recombinant proteins expressed in insoluble fractions of bacteria, led to a mere 50% of the total protein being correctly refolded. Separation onto a gel filtration column led to two pools: one active and the other not . These two fractions could not be distinguished from one another using these analytical approaches.…”

“…Roughly half a decade ago, we decided to enter a series of programs aimed at synthesizing a series of proteins of growing length from ubiquitin and calstabin to quinone reductase (which contains 226 amino acids and with which we failed). The difficulties encountered in developing a universal methodology to obtain such proteins, most of which were enzymes, led us to limit ourselves to ~200 amino acids.…”

On the Organization of a Drug Discovery Platform

“…For this reason, we chose to install a dedicated laboratory inside a synchrotron (Soleil, St. Aubin, France) to gain full access not only to the beamlines but also to the vast scientific community behind this instrument. This choice led to several collaborative academic-oriented lines of research [136,141] and to more internal research programs. In preparing the next steps of crystallization-the step involving the receptor and more generally membrane proteins-we assessed a series of methodologies for the solubilization and purification of functionally competent receptors [56,57].…”

“…In doing so, we learned two important things: (1) such an approach is feasible, even though it led to small quantities of material, at least to start with, and the material was indistinguishable from the recombinant version and (2) the apparent mono-peak analysis of the result of the synthesis revealed, after further analyses, that the protein could be separated in two distinct peaks, one a fully refolded protein with catalytic activity and the other a denatured protein with no catalytic activity and no refolding [141]. These findings led us to consider that large peptides/proteins that are chemically synthesized and often described as being used in the literature (e.g., EPO, growth factors) might be a mixture of active and inactive substances if not appropriately characterized.…”

“…In doing so, we not only identified and incorporated strong scientists but also could assign new technologies to categories such as 'dream', 'usable soon', 'needs more time', etc. Among the discoveries that we have published are the ligase use for in cellulo transformation of proteins [163], including antibodies; cellular imaging using large synchrotron instruments; synthetic proteins that can be as active as the recombinants [141]; and synthesis of proteins and peptides modified by ubiquitination the way it happens in the cell (as opposed to what was commercially available at that time) [115]. In addition, we started our first trials in thermodynamics, structural biology, new crystallization techniques, native mass spectrometry for structural biology approaches [152] and for target-molecule relationships, and the use of human stem cells as an alternative to rat cardiomyocytes in first culture, among other initiatives.…”