ABSTRACT
Quantum dots (QDs) can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution (super-resolution fluorescence imaging). However, the toxicity of Cd in the prototypical CdSe-based QDs can limit their use in biological applications. Furthermore, commercial CdSe QDs are usually modified with relatively thick shells of both inorganic and organic materials to render them in the 10-20 nm size range, which is relatively large for biological labels. In this report, we present compact (4-6 nm) CuInS2/ZnS (CIS/ZnS) and compare them to commercially sourced CdSe/ZnS QDs for their blinking behavior, localization precision and super-resolution imaging. Although commercial CdSe/ZnS QDs are brighter than the more compact Cd-free CIS/ZnS QD, both give comparable results of 4.5-5.0-fold improvement in imaging resolution over conventional TIRF imaging of actin filaments. This likely results from the fact that CIS/ZnS QDs show very short on-times and long off times which leads to less overlap in the point spread functions of emitting CIS/ZnS QD labels on the actin filaments at the same labeling density. These results demonstrate that CIS/ZnS QDs are an excellent candidate to complement and perhaps even replace the larger and more toxic CdSe-based QDs for robust single- molecule super-resolution imaging.
ABSTRACT
Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for the LHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.
