Site-specific protein modification: advances and applications

https://doi.org/10.1016/j.cbpa.2006.11.036Get rights and content

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.

Introduction

Although methods to acquire whole-genome sequences have become routine, analysis of these data indicates that the bulk of the genomic content encodes putative proteins with unknown function. Realization that the complexity of this body of information cannot be analyzed by current computational and genomic methods has fueled the development of new techniques for functional characterization. Post-translational modifications, which have a vital role in modulating in vivo activity, complicate these investigations, so that traditional profiling techniques will not be able to arrive at a comprehensive understanding of this material. Elucidation of modification pathways, as a means to better understand these mechanisms of activation and regulation, has bred a field of scientists conducting interdisciplinary studies performing chemical transformations in biological contexts to solve these elusive problems. These studies have led to the discovery of biochemical systems with exquisite sequence specificity and, through delicate probing of their chemical selectivity, have identified permissive substrate analogues with functionality unperturbed by cellular chemistries. Because of their exemption from cellular metabolism, these analogues can install uniquely reactive moieties to the target protein upon modification, and these can be detected by fluorescent visualization or isolation, thus enabling insight into cellular localization of protein populations that bear such derivatizations. The robust nature of these techniques has encouraged their application to additional biological problems, and the orthogonality of these probes to cellular processes has further extended their use from proteomic analysis and genomic annotation to the imaging of biochemical processes at the cellular level.

In this review, we focus on recent advances in the biochemical and proteomic fields toward the ability to label proteins site-specifically, as well as on new applications of post-translational modification toward cellular imaging. Being an abridged summary, this is by no means a substitute for other recent discussions on either activity-based protein-profiling [1••] or tools for cellular imaging [2••], it does not span the scope of engineered systems that use overt knowledge of the protein system (exemplified in [3]), and it does not cover material that was adequately described previously [4, 5]. Herein, as a continuation of this annual series, we discuss recent advances in the site-specific labeling of proteins that are of interest to the chemical biology community.

Section snippets

Biochemical (in vitro) tools

General techniques for the in vitro chemical labeling of native (non-fusion) proteins have been used to date, but they are becoming less applicable, in part because of the reactivity limitations of the 20 proteinogenic amino acids. These methods use selective chemistries that exploit solvent-accessible thiols (of cysteine residues), amines of lysine residues and the N-terminus) and, more recently, carboxylates (of aspartate, glutamate and the C-terminus) [6]. Although these groups can be

Proteomic (ex vivo) tools

In the proteomic arena, exhaustive effort has been put forth to develop new techniques that enable the identification of post-translationally modified proteins. More than 200 nontemplated alterations of protein surfaces are known to occur [13], and these have important roles in enzyme regulation, signal transduction, protein half-life and subcellular localization. Of these, phosphorylation is of particular interest to researchers because of its ubiquitous nature, playing a pivotal role in

Cellular imaging and recombinant tools

Post-translational modifications have also been investigated as potential new avenues for the imaging of cellular processes. The enzymes that govern some of these processes show stringent sequence requirements regarding their proteinacious substrates but are tolerant of derivatizations made to the small-molecule undergoing ligation: such characteristics make them ideal for in vivo applications (Table 1). Studies to minimize recognition motifs have led to the shortening of these polypeptide

Conclusions

Site-specific labeling of proteins with small molecules promises to advance our understanding of biological systems through several pathways. Newly developed techniques will provide protein chemists with the ability to prepare homogeneous substrate pools for biophysical studies. Permissive substrate analogues to analyze modification events will increase our understanding of these key modes of activity modulation. Advanced computational assignment of the putative function of gene products will

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

References (39)

  • Y. Takaoka et al.

    One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity

    J Am Chem Soc

    (2006)
  • I. Chen et al.

    Site-specific labeling of proteins with small molecules in live cells

    Curr Opin Biotechnol

    (2005)
  • L.W. Miller et al.

    Selective chemical labeling of proteins in living cells

    Curr Opin Chem Biol

    (2005)
  • J.M. Gilmore et al.

    N-terminal protein modification through a biomimetic transamination reaction

    Angew Chem Int Ed Engl

    (2006)
  • J. Xie et al.

    A chemical toolkit for proteins – an expanded genetic code

    Nat Rev Mol Cell Biol

    (2006)
  • J.W. Chin et al.

    An expanded eukaryotic genetic code

    Science

    (2003)
  • Z. Zhang et al.

    Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

    Proc Natl Acad Sci USA

    (2004)
  • S.C.B. Yan et al.

    Post-translational modifications of proteins – some problems left to solve

    Trends Biochem Sci

    (1989)
  • J.J. Allen et al.

    Bio-orthogonal affinity purification of direct kinase substrates

    J Am Chem Soc

    (2005)
  • Cited by (0)

    View full text