Photoaffinity labeling

Last updated

Photoaffinity labeling is a chemoproteomics technique used to attach "labels" to the active site of a large molecule, especially a protein. The "label" attaches to the molecule loosely and reversibly, and has an inactive site which can be converted using photolysis into a highly reactive form, which causes the label to bind more permanently to the large molecule via a covalent bond. [1] [2] The technique was first described in the 1970s. [3] Molecules that have been used as labels in this process are often analogs of complex molecules, in which certain functional groups are replaced with a photoreactive group, such as an azide, a diazirine or a benzophenone. [4] [5] [6]

Related Research Articles

Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology.

<span class="mw-page-title-main">William Lipscomb</span> American chemist (1919–2011)

William Nunn Lipscomb Jr. was a Nobel Prize-winning American inorganic and organic chemist working in nuclear magnetic resonance, theoretical chemistry, boron chemistry, and biochemistry.

Combinatorial chemistry comprises chemical synthetic methods that make it possible to prepare a large number of compounds in a single process. These compound libraries can be made as mixtures, sets of individual compounds or chemical structures generated by computer software. Combinatorial chemistry can be used for the synthesis of small molecules and for peptides.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

Juvenile hormones (JHs) are a group of acyclic sesquiterpenoids that regulate many aspects of insect physiology. The first discovery of a JH was by Vincent Wigglesworth. JHs regulate development, reproduction, diapause, and polyphenisms. The chemical formula for juvenile hormone is .

In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin. Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

<span class="mw-page-title-main">Immunoassay</span> Biochemical test for a protein or other molecule using an antibody

An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.

In chemistry, a nitrene or imene is the nitrogen analogue of a carbene. The nitrogen atom is uncharged and univalent, so it has only 6 electrons in its valence level—two covalent bonded and four non-bonded electrons. It is therefore considered an electrophile due to the unsatisfied octet. A nitrene is a reactive intermediate and is involved in many chemical reactions. The simplest nitrene, HN, is called imidogen, and that term is sometimes used as a synonym for the nitrene class.

Chemical biology is a scientific discipline between the fields of chemistry and biology. The discipline involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. In contrast to biochemistry, which involves the study of the chemistry of biomolecules and regulation of biochemical pathways within and between cells, chemical biology deals with chemistry applied to biology.

Affinity labels are a class of enzyme inhibitors that covalently bind to their target causing its inactivation. The hallmark of an affinity label is the use of a targeting moiety to specifically and reversibly deliver a weakly reactive group to the enzyme that irreversibly binds to an amino acid residue. The targeting portion of the label often resembles the enzyme's natural substrate so that a similar mode of noncovalent binding is used prior to the covalent linkage. Their usefulness in medicine can be limited by the specificity of the first noncovalent binding step whereas indiscriminate action can be utilized for purposes such as affinity labeling - a technique for the validation of substrate-specific binding of compounds.

DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells.

<span class="mw-page-title-main">Systematic evolution of ligands by exponential enrichment</span> Technique for producing oligonucleotides that specifically bind to a target

Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.

<span class="mw-page-title-main">Meir Wilchek</span> Israeli biochemist (born 1935)

Meir Wilchek is an Israeli biochemist. He is a professor at the Weizmann Institute of Science.

Alice Yen-Ping Ting is Taiwanese-born American chemist. She is a professor of Genetics, of Biology, and by courtesy, of Chemistry at Stanford University. She is also a Chan Zuckerberg Biohub investigator and a member of the National Academy of Sciences.

Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes.

DNA-encoded chemical libraries (DEL) is a technology for the synthesis and screening on an unprecedented scale of collections of small molecule compounds. DEL is used in medicinal chemistry to bridge the fields of combinatorial chemistry and molecular biology. The aim of DEL technology is to accelerate the drug discovery process and in particular early phase discovery activities such as target validation and hit identification.

In organic chemistry, diazirines are a class of organic molecules consisting of a carbon bound to two nitrogen atoms, which are double-bonded to each other, forming a cyclopropene-like ring, 3H-diazirene. They are isomeric with diazocarbon groups, and like them can serve as precursors for carbenes by loss of a molecule of dinitrogen. For example, irradiation of diazirines with ultraviolet light leads to carbene insertion into various C−H, N−H, and O−H bonds. Hence, diazirines have grown in popularity as small, photo-reactive, crosslinking reagents. They are often used in photoaffinity labeling studies to observe a variety of interactions, including ligand-receptor, ligand-enzyme, protein-protein, and protein-nucleic acid interactions.

PRIME is a molecular biology research tool developed by Alice Y. Ting and the Ting Lab at MIT for site-specific labeling of proteins in living cells with chemical probes. Probes often have useful biophysical properties, such as fluorescence, and allow imaging of proteins. Ultimately, PRIME enables scientists to study functions of specific proteins of interest.

Chemoproteomics entails a broad array of techniques used to identify and interrogate protein-small molecule interactions. Chemoproteomics complements phenotypic drug discovery, a paradigm that aims to discover lead compounds on the basis of alleviating a disease phenotype, as opposed to target-based drug discovery, in which lead compounds are designed to interact with predetermined disease-driving biological targets. As phenotypic drug discovery assays do not provide confirmation of a compound's mechanism of action, chemoproteomics provides valuable follow-up strategies to narrow down potential targets and eventually validate a molecule's mechanism of action. Chemoproteomics also attempts to address the inherent challenge of drug promiscuity in small molecule drug discovery by analyzing protein-small molecule interactions on a proteome-wide scale. A major goal of chemoproteomics is to characterize the interactome of drug candidates to gain insight into mechanisms of off-target toxicity and polypharmacology.

References

  1. Photoaffinity labeling
  2. Photoaffinity labeling, Gold Book
  3. Ruoho, A. E.; Kiefer, H.; Roeder, P. E.; Singer, S. J. (1973). "The mechanism of photoaffinity labeling". Proceedings of the National Academy of Sciences of the United States of America. 70 (9): 2567–2571. Bibcode:1973PNAS...70.2567R. doi: 10.1073/pnas.70.9.2567 . PMC   427057 . PMID   4517671.
  4. Bush, J. T.; Walport, L. J.; McGouran, J. F.; Leung, I. K. H.; Berridge, G. (2013). "The Ugi four-component reaction enables expedient synthesis and comparison of photoaffinity probes". Chemical Science. 4 (12): 4115–4120. doi: 10.1039/C3SC51708J .
  5. Panov, M. S.; Voskresenska, V. D.; Ryazantsev, M. N.; Tarnovsky, A. N.; Wilson, R. M. (2013). "5-Azido-2-aminopyridine, a New Nitrene/Nitrenium Ion Photoaffinity Labeling Agent That Exhibits Reversible Intersystem Crossing between Singlet and Triplet Nitrenes". Journal of the American Chemical Society. 135 (51): 19167–19179. doi:10.1021/ja405637b. PMID   24219134.
  6. Akiyama, S.; Cornwell, M. M.; Kuwano, M.; Pastan, I.; Gottesman, M. M. (1988). "Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog". Molecular Pharmacology. 33 (2): 144–147. PMID   2893251.