Membrane Protein Stoichiometry Determined from the Step‐Wise Photobleaching of Dye‐Labelled Subunits

Many important proteins function as multimeric complexes, which often contain different types and numbers of subunits. Such proteins play a key role in a wide variety of cellular events, including signal transduction, cell regulation, transport and energy generation. Protein oligomers are associated with many human diseases including early onset Parkinson’s, Alzheimer’s and Huntington’s disease. Such complexes also play key roles in the pathogenic action of many bacterial toxins. If we are to understand the widespread roles of protein complexes, additional methods must be developed that are capable of measuring the temporal variations in subunit stoichiometry that must occur during complex assembly. Several techniques provide information on protein stoichiometry. The mapping of high resolution X-ray crystal structures onto lower resolution cryoelectron microscopy images provides one method for determining high-resolution structures of large complexes. 6] The difficulty of this sophisticated approach, however, is reflected in the limited number of publications successfully combining both techniques. Atomic force microscopy (AFM) can also be used to determine subunit stoichiometry either directly or by specifically tagging subunits with other large molecules. Other techniques capable of providing information on the stoichiometry of protein complexes include analytical ultracentrifugation, gel electrophoresis, Fcrster resonance energy transfer (FRET) and mass spectrometry. These methods vary greatly in both experimental complexity and their utility for subunit determination. For example—although costly and time consuming—cryoelectron microscopy and X-ray crystallography are capable of providing exquisite resolution of large protein complexes. On the other hand, FRET only requires judicious chemical modification of two proteins, but is limited to studying the interactions of two (or possibly three) partners in what could be a much larger complex. Single-molecule fluorescence (SMF) microscopy is a relatively new technique capable of resolving the stoichiometry of a fluorescently labelled protein complex. SMF also has the potential to measure the dynamics of protein oligomerisation both in vitro and in vivo, without the need to immobilise the protein on a surface or within a crystal. SMF is not able to provide a structure of an individual molecule (as with AFM or cryoelectron microscopy), however, SMF can be used to visualise molecular complexes tagged with different fluorescent labels. One SMF method capable of resolving subunit stoichiometry is simply to count the number of photobleaching steps within a single complex. Photobleaching is the irreversible loss of fluorescence in a molecule due to changes in its structure following a light-induced chemical reaction. For an individual fluorophore, photobleaching is observed as a step change in fluorescence intensity. If each subunit within a protein complex is labelled with a fluorescent molecule, the total number of photobleaching steps determines the number of subunits within the complex. Multiple photobleaching steps have been observed in a randomly labelled mRNA–ribosome complex and in fluorescently labelled polymers. Photobleaching steps have also been used to determine the number of fluorescently labelled proteins encapsulated within a lipid vesicle, to infer the stoichiometry of stator components within the bacterial flagellar motor and to determine the number of labels on a green fluorescent protein dimer. Here, we employ the stepwise changes in SMF intensity during photobleaching to determine the number of subunits within a membrane–protein complex. b-Barrel pore-forming toxins (b-PFTs) are a class of multimeric membrane proteins that assemble on lipid membranes to form bilayer-spanning pores. Their known stoichiometries vary from small oligomers to large 30–50 subunit complexes. a-Hemolysin (aHL) is the archetypal b-barrel-forming toxin. High-resolution crystallographic, AFM and biochemical studies suggests that aHL forms a heptameric pore. Other AFM experiments have reported hexameric aHL, thus indicating that some variation in subunit stoichiometry might be possible. Leukocidin is a bicomponent b-PFT with significant sequence homology to aHL. It is formed from two different polypeptide subunits, LukF and LukS, both of which have several different isoforms. The structures of both LukS and LukF monomers have been resolved with X-ray crystallography. 30] However, in contrast to aHL, there is much less consensus regarding the structure of the fully assembled leukocidin pore. No crystal structure of the pore complex is available, and the numbers of each component are not conclusively known. Biochemical cross-linking and chemical modification during single-channel recording have provided evidence that leukocidin is an octameric pore with an alternating arrangement of subunits. Other experiments indicate that leukocidin forms pores with alternative stoichiometries: either hexameric or heptameric. Single-molecule FRET experiments have also been performed with leukocidin. Higuchi and co-workers [a] Dr. S. Cheley, Dr. M. I. Wallace, Prof. Dr. H. Bayley Department of Chemistry, University of Oxford Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+1)44-1865-285002 [b] Dr. S. K. Das, Dr. M. Darshi Department of Molecular & Cellular Medicine, The Texas A&M University System Health Science Center College Station, TX 77843-1114 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author: representative image showing individually resolved aHL complexes on a glass cover-slip surface, and a graph showing how the intensity of each single-molecule spot correlates with the number of photobleaching steps detected.