2Institute for Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany
3Department of Biology and Physics, Wilkes University, Wilkes-Barre, Pennsylvania, USA
K.V.G.'s present address is the Institute for Chemistry and Biochemistry, Free University of Berlin, Takustr. 6, 14195 Berlin, Germany.
Here we describe a labeling technique for the covalent linkage of quantum dots to transmembrane receptors for single-molecule tracking. Our method combines the acyl carrier protein (ACP) technique with coenzyme A (CoA)–functionalized quantum dots to covalently attach quantum dots to ACP fusions of receptor proteins. The advantages of this approach include: (i) the use of a smaller attachment linker than in many other quantum dot–labeling systems; (ii) the ability to achieve a reliable 1:1 fluorophore-to-receptor labeling stoichiometry; (iii) the specificity of the method; and (iv) the covalent nature of the quantum dot linkage. We demonstrate the general suitability of this technique in single-molecule tracking, internalization, and trafficking studies by imaging two different transmembrane receptors in living cells.
Detection of single fluorescently labeled proteins is useful in determining their local mobility, local stoichiometry, fundamental molecular interactions and molecular function. Numerous single-molecule studies have demonstrated the unique functions of proteins, revealing both large and subtle details of their biomolecular interactions. Two key issues with such studies are the biochemical method of fluorophore attachment and the proper choice and use of the fluorophore itself (for reviews, see References 1-4).
For analysis of the molecular mobility of membrane receptors, specific localization along with the correct determination of the motion of the fluorescently labeled receptor requires high signal-to-background ratios and acquisition rates and a rigid attachment of a minimized fluorophore (5,6). Fluorescently labeled receptors and ion channels for single-molecule studies on living cells can be obtained with fluorescent fusion proteins (7-10), by direct covalent bonding of fluorophores to cysteine or amino reactive groups, or with protein tags that react with modified f luorophores (11). However, while these techniques are well-established for standard organic fluorophores, general schemes for direct labeling by quantum dots (QDs) are still in their infancy (1-4).
Single-molecule tracking with QDs displays superior performance over tracking with large latex beads since QDs are ~10× smaller. In addition, they typically yield higher fluorescence signals than standard organic dyes and fluorescent proteins due to their improved brightness and resistance to photobleaching. With the recent advent of small, noncommercially available QDs, the size limitations of standard commercial QDs have been overcome, making them comparable in size to fluorescent proteins and only somewhat larger than the brightest fluorescing organic dyes (12,13).
QD tracking has been limited in the number of available labeling techniques. To date, most QDs for tracking are conjugated to antibodies, which unless they are directly conjugated to primary Fab fragments, can increase the effective radius of the QD by ≥10 nm (1,3,4). Although tracking receptors with QD-conjugated secondary antibodies has been successful (1,3), the desire to reduce the common drawbacks of antibody labeling (14-17) has led to new direct QD binding techniques.
Direct attachment of QDs to receptors can be achieved by normal amino or cysteine nonspecific reactions (1-4). Another possibility is the biotinylation of the receptor and addition of either streptavidin-coated QDs or QDs conjugated with the recently generated m-streptavidin of only 159 amino acid residues (18,19). Yet another method is to covalently link a QD to a fusion protein containing a HaloTag, a modified haloalkane dehydrogensase of 296 amino acids, which can covalently bind a modified chloroalkane-labeled QD (20,21).
Many other labeling systems are useful for imaging and biological quantification of proteins (22-24); however, with the exception of the aformentioned techniques, they are either noncovalent or lack suitable protocols for QD labeling. Some systems—including FlAsH (25), hexahistidine (13,26,27), biotin acceptor peptide (28), and Q-tag (29)—are so stable with organic dyes that they have been demonstrated with single-molecule detection.
The acyl carrier protein (ACP) system is especially well-suited for the specific labeling and tracking of single receptors on living cells (30). With only 77 amino acids, ACP is relatively smaller than comparable tags such as the SNAP and CLIC tags (22-24) and has reliable reactivity to coenzyme A (CoA) bound to organic fluorophores or to biotin (30) in a reaction catalyzed by phosphopantetheinyl transferase (PPTase). The advantages of tracking with ACP-CoA and organic f luorophores were demonstrated on G protein–coupled receptors (GPCRs) (9,10), and AMPA receptors (31). In particular, tracking with ACP is advantageous since the receptors fused to ACP retain biological activity. Furthermore, the covalent binding of the CoA fluorophore by ACP is specific and efficient. The same system was also used for internalization studies (9,10,31). However, until now, protocols for the direct attachment of QDs to receptors via an ACP tag did not exist.
Here we introduce a method for labeling ACP tags with QDs covalently bound to CoA in a 1:1 ratio. The QD:CoA covalent binding occurs via straightforward thiol-reactive chemistry in an appropriate concentration of amine-blocking groups on the QDs. We demonstrate the successful labeling of two different receptors: the parathyroid hormone receptor (PTHR), which is a GPCR, and the bone morphogenetic protein type II receptor (BRII). We compare the labeling and tracking of both receptors with either our ACP-CoA-QD technique or antibody QD conjugates. We find that ACP-CoA-QD labeling enables more precise tracking, longer tracking times, and the possibility to determine all stages in the fate of individual receptors, including behavior at the membrane, internalization, and post-internalization trafficking events, such as recycling. To our knowledge, this technique provides the shortest specific covalent tether of a QD with a receptor.