Abstract
Proteins constitute the majority of nature’s worker biomolecules. Designed for specific functions, complex tertiary structures make proteins ideal candidates for analysing natural systems and creating novel biological tools. Owing to both their large size and the need for proper folding, de novo synthesis of proteins has been quite a challenge, leading scientists to focus on modifying protein templates already provided by nature. Recently developed methods for protein modification fall into two broad categories: those that can modify the natural protein template directly and those that require genetic manipulation of the amino acid sequence before modification. The goal of this Review is not only to provide a window through which to view the many opportunities created by novel protein modification techniques‚ but also to act as an initial guide to help scientists find direction and form ideas in an ever-growing field. In addition to highlighting methods reported in the past 5 years, we aim to provide a broader sense of the goals and outcomes of protein modification and bioconjugation in general. While the main body of this paper comprises reactions involving the direct modification of expressed proteins, some further functionalization strategies as well as biological applications are also acknowledged. The discussion concludes by speculating which trends and discoveries will most likely come next in the field.
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Acknowledgements
The authors thank the Foundation for Science and Technology (FCT) Portugal (FCT Investigator to G.J.L.B., IF/00624/2015 and Postdoctoral Fellowship to P.M.S.D.C.), the Herchel Smith Fund (PhD Studentship to E.A.H.) and the European Union Horizon 2020 programme (Marie Skłodowska-Curie ITN grant agreement no. 675007 to G.J.L.B. and Marie Skłodowska-Curie IEF grant agreement no. 702574 to B.L.O.) for funding. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF\R\180019) and the recipient of a European Research Council Starting Grant (TagIt, grant agreement no. 676832).
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All authors made substantial contributions to the discussion and organization of the content as well as reviewed and/or edited the manuscript before submission. Additionally, E.A.H. conducted the research, wrote the main body of the paper, edited the figures and put the complete manuscript together; P.M.S.D.C. played an integral role in writing the introduction, researching and providing content guidance at all stages of manuscript preparation; and B.L.O. designed and created the figures. G.J.L.B. coordinated the research and writing.
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Glossary
- Post-translational modification
-
(PTM). A post-translational, covalent protein modification that has critical roles in cell signalling and control of protein activation or function.
- Site-selective
-
Referring to modification methods that target a certain residue over other types of amino acids.
- Homogeneous product
-
A single product — type and position of modification — is formed. Conversely, heterogeneous products are those in which different and multiple modifications have occurred, including on different amino acids or at various occurrences of the same amino acid type.
- Site-specific
-
Referring to modification methods that target a single occurrence of a particular type of amino acid.
- Protein microenvironment
-
The manipulation of amino acid side chain properties (for example, steric or electric characteristics) and reactivity based on the identity of surrounding amino acids in the protein sequence.
- Human insulin
-
A protein that is made up of two separate chains of amino acids labelled A and B, bound together by two disulfide bridges.
- Canonical amino acids
-
The standard 20 amino acid types encoded and inserted naturally by the genetic code and by native protein biosynthesis systems.
- SH3-domain proteins
-
Proteins that contain SH3 domains for the regulation of cytoplasmic signalling pathways.
- Antibodies
-
Proteins that are composed of two main regions: Fc regions (constant regions for the support and stability of the antibody) and Fab regions (variable regions of the antibody that must be preserved in order to retain affinity and specificity for a corresponding antigen).
- Disulfide rebridging
-
A process by which two Cys residues, revealed by disulfide reduction, reform the disrupted disulfide either through the construction of a mixed disulfide or through the introduction of a synthetic stapling molecule to connect the two residues.
- Conjugate payload
-
The chemical linker and added functionality (for example, fluorophore or cytotoxic drug) in a protein conjugate.
- Noncanonical amino acids
-
(ncAAs). Amino acids that are most often synthesized and non-proteinogenic (with the exception of Sec and Pyl) and can be inserted either residue-specifically or site-specifically into protein sequences.
- Endogenous residues
-
Amino acid residues that are synthesized by the host organism rather than by artificial synthesis.
- Click chemistry
-
Chemical reactions that can be defined by high reaction and conversion rate, green solvent systems, low by-product levels and broad functional group applicability.
- Bioorthogonal reactions
-
Chemical reactions that can be executed in the complex environment of living systems (that is, in the presence of many nucleophiles, reductants and so on) without altering or affecting native processes.
- Orthogonal aminoacyl-tRNA synthetase–tRNA pairs
-
These orthogonal pairs can use native protein biosynthesis machinery for the site-specific insertion of noncanonical amino acids and require that no native RNA synthetase be able to aminoacylate the incorporated tRNA and no native tRNA be modified by the incorporated RS.
- Fusion proteins
-
Proteins that are produced by combining parts from different proteins or proteins with smaller amino acid sequences/tags to create one expressed entity.
- Upconversion nanoparticles
-
Nanoscale particles that allow for photon upconversion (the absorption of two lower-energy photons to create one higher-energy, emitted photon) for imaging and sensors in deep tissue environments.
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Hoyt, E.A., Cal, P.M.S.D., Oliveira, B.L. et al. Contemporary approaches to site-selective protein modification. Nat Rev Chem 3, 147–171 (2019). https://doi.org/10.1038/s41570-019-0079-1
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DOI: https://doi.org/10.1038/s41570-019-0079-1
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