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April 01, 2008 (Vol. 28, No. 7)

Studying Protein Interactions in Living Cells

Method for Protein Crosslinking Utilizes Photoreactive Amino Acids

  • Proteins are the building blocks of all organisms, and protein-protein interactions are the foundation for cellular structure, metabolism, and communication. Protein-protein interactions range from contacts of protein-complex subunits to dynamic connections among enzymatic signaling networks.

    Two common techniques to study protein interactions include GST pull-downs and coimmunoprecipitations. Both methods, however, analyze proteins following cell lysis, which typically involves detergents that may cause aberrant protein interactions by disrupting hydrophobic interfaces of protein complexes or by mixing of cellular compartments.

    Chemical crosslinking of protein complexes in live cells is a powerful way to capture both stable and transient protein-protein interactions in their native environment. Traditional amine-reactive crosslinkers such as formaldehyde or NHS-ester derivatives have been used to study protein-protein interactions in vitro but have varying degrees of success for studying protein complexes in their native environment.

    Formaldehyde has been used extensively to reversibly crosslink proteins through primary amines with a zero-length bond distance. It, however, spontaneously forms polymers in solution and is not protein-specific as it also crosslinks proteins to DNA and other macromolecules.

    Bifunctional NHS-ester crosslinkers (e.g., DSG and DSS) are more protein-specific than formaldehyde but only crosslink specific amino acid residues at defined spacer lengths. Additionally, formaldehyde and NHS esters must permeate cell membranes in order to crosslink intracellular proteins and must be quenched to prevent excessive crosslinking.

  • Click Image To Enlarge +
    Figure 1

    A new method for protein crosslinking in live cells involves metabolically incorporating photoreactive amino acids during protein expression. Thermo Fisher Scientific has synthesized two unique photoreactive amino acid derivatives, Photo-Leucine and Photo-Methionine, that have similar structures to their respective natural amino acids (Figure 1).

  • Unlike some amino acid derivatives, these photoreactive analogs are recognized by the endogenous, mammalian protein-synthesis machinery and do not require any genetic manipulation of tRNAs or tRNA acetyl transferases to be incorporated directly into proteins.

    Photocrosslinking Using Photoreactive Amino Acids

    Photo-Leucine and Photo-Methionine are amino acid derivatives that contain a diazirine moiety for ultraviolet (UV) photocrosslinking of proteins. Photoactivation of diazirines with UV light creates a reactive carbene intermediate that irreversibly crosslinks proteins at any amino acid side chain or peptide backbone with a zero-length bond distance. These compounds allow the researcher to use UV light to irreversibly photocrosslink proteins within protein-interaction domains containing methionine and/or leucine.

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