Novel protein science enabled by total chemical synthesis

Abstract

Chemical synthesis is a well-established method for the preparation in the research laboratory of multiple-tens-of-milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d-protein and l-protein enantiomers facilitate crystallization and determination of protein structures by X-ray diffraction. d-Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d-protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear-loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time-resolved FTIR for the elucidation of protein structure-activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein-inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d-protein forms of polymerase enzymes.

Keywords: mirror image proteins; native chemical ligation; polypeptide chain topology; racemic protein crystallography; site-specific labeling; total chemical synthesis.

Figure 1

Synthesis and characterization of a synthetic protein. (A) General synthetic scheme. Unprotected peptide segments, prepared by solid phase peptide synthesis (SPPS) are condensed by (native) chemical ligation to give the full‐length polypeptide chain which is folded to give the protein molecule. (B) Direct infusion electrospray mass spectrum of [Lys24, 38, 83]EPO prepared by total chemical synthesis. Each of the major peaks corresponds to a different charge state (number of excess protons) of the synthetic protein molecule. Note the absence of minor peaks on the low m/z side of each major peak, showing the absence of microheterogeneity in the synthetic protein product. (The minor peaks on the high m/z side of each peak are Na⁺ and other metal ion adducts.)

Figure 2

Racemic protein crystallography. Adapted from Reference 29.

Figure 3

Structure of the protein Rv1738 from M. tuberculosis determined by racemic protein crystallography. (A) Racemic crystal. (B) X‐ray diffraction pattern. (C) Mirror image structures of the Rv1738 homodimer. The blue image is the l‐homodimer and the red image is the d‐homodimer. The inversion center is shown as a cyan dot. (D) 2Fo–Fc electron density map for a Trp side chain. Adapted from Ref. 31.

Figure 4

Screening a chiral natural product library against the mirror image forms of an enzyme can provide novel hits. Synthesis of the mirror image of a hit compound then gives a novel natural product‐related molecule active against the native enzyme.

Figure 5

Quasi‐racemic X‐ray crystallography determination of the structure of an interpenetrating linear‐loop topological analogue of crambin. (A) Crystallographic unit cell. (B) Cartoon representation of the backbone structure. The new amide link is shown as CPK spheres. C. electron density 2Fo–Fc map with the fitted structure of the new amide bond shown as sticks. (D) Superimposition of the crambin topological analogue structure (green) and the structure of native crambin (cyan) generated by inverting the structure of d‐crambin. Both protein structures were ontained in the same experiment. Adapted from Ref. 34.

Figure 6

EPO glycoforms prepared by total chemical synthesis. (A) Modular convergent synthetic scheme. The bottom row shows cartoon representations of the five glycoforms. (B) Direct infusion electrospray mass spectra of the synthetic glycoproteins. Observed masses of the synthetic glycoproteins were 20,540 Da, 22,746 Da (three isomeric glycoforms), and 24,952 Da. Adapted from Ref. 97.

Figure 7

X‐ray structure of an a priori designed protein molecule. The circular polypeptide chain is shown in purple with cartoon representation of the helical regions. The aromatic ring used to covalently join the three helices and constrain the folded structure is shown in cyan.102

Figure 8

Measurement of distances in the voltage gated sodium channel Nav1.1 protein molecule. Lanthanide resonance energy transfer (LRET) measurements were enabled by the total chemical synthesis of small protein toxins site‐specifically labeled with a fluorophore dye. Adapted from Ref. 110.