We have generated a monovalent streptavidin, by making a chimeric streptavidin tetramer with a single active biotin binding subunit. This monovalent streptavidin bound to biotin with similar affinity and stability as wild-type streptavidin, did not rearrange its subunits over time and had high thermostability. Monovalent streptavidin should permit one to make use of its femtomolar binding affinity without additional unwanted multimerization.
Aside from illustrating how monovalent streptavidin can be used for stable and cross-linking–free protein detection in cellular imaging experiments, our experiments with neuroligin-1 demonstrate how engineered streptavidins can be used to examine the biological consequences of controlled clustering of cellular proteins. Cross-linking is used by cells to regulate protein activity, mobility and interactions, as demonstrated for many growth factor receptors and transcription factors8
. Here we only compared monovalent and tetravalent (wild-type) streptavidins, but it would also be possible to use the entire series of purified streptavidin heterotetramers to examine the functional effects of receptor dimerization, trimerization and tetramerization.
The many alternative elegant approaches to site-specific labeling of proteins in living cells cannot all be described here, but have recently been reviewed17–19
. The key point is that no single labeling approach is ideal in every circumstance. For example, fluorescent proteins or biarsenical labeling of tetracysteine tags20
are valuable methods that do not suffer from probe dissociation or cause cross-linking, but they are limited for single-particle tracking by the moderate brightness and photobleaching of their fluorophores. Biarsenical labeling also cannot be used to label proteins specifically at the cell surface, although it has the advantage over our method that it can be used to label intracellular proteins. The advantages of labeling by means of site-specific biotinylation and monovalent streptavidin, as compared to antibody labeling, are twofold. First, the biotin-streptavidin interaction provides a much more stable linkage that permits long-term imaging over hours or even days. This strong interaction also eliminates the concern that the label might rapidly dissociate and reassociate with different cell-surface proteins over the time course of the imaging experiment, complicating tracking measurements. Second, antibodies are bivalent and can cross-link target proteins, as wild-type streptavidin does. Fab antibody fragments bind monovalently, but usually have substantially lower effective affinities, compounding the problem of antibody-epitope instability. Conversely, when high-affinity antibodies are available, antibodies have the advantage that they can be used to detect endogenous proteins, avoiding possible artifacts from trans-gene overexpression.
The need to purify different chimeric forms of streptavidin, as previously performed to improve reversibility21,22
and as shown here, could be avoided if the four subunits could be genetically joined to make a single-chain streptavidin. But the large distance between the termini of streptavidin means that long linkers would be required, which are likely to impair folding. Attempts to circumvent this problem by circularly permuting streptavidin have yielded forms with Kd
. A circularly permuted tetravalent single-chain avidin with wild-type binding affinity was recently reported25
. It will be valuable to generate an analogous single-chain tetravalent streptavidin and then to make it monovalent using the approach presented here, as avidin binds ~40-fold less tightly to biotin conjugates than streptavidin26
Bottom-up nanotechnology refers to the emerging use of self-assembly to construct multimolecular assemblies on the nanometer scale27
. Streptavidin is one of the most common bridges in such assemblies, linking biotinylated DNA molecules, proteins and inorganic structures including carbon nanotubes and gold particles27
. Such streptavidin bridges are either four-way junctions, if the biotinylated ligand is used at a saturating concentration, or statistical mixtures of zero- to four-way junctions, if the biotinylated ligand is used at a subsaturating concentration. The use of the chimeric streptavidin tetramers described in this paper should permit one to select whether a one-, two-, three- or four-way junction is generated. Poor yields of the desired junction, resulting from using a statistical mixture, are tolerable if there is only one step in assembly, but give unacceptable final yields if there are multiple steps in the assembly. This limits the complexity of the nanostructure that can be assembled. Thus chimeric streptavidins should be valuable building blocks for the construction of novel microarrayed sensors28
and microelectronic circuits29