Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot

Abstract

Quantum dots (QDs) possess highly desirable optical properties that make them ideal fluorescent labels for studying the dynamic behavior of proteins. However, a lack of characterization methods for reliably determining protein-quantum dot conjugate stoichiometry and functionality has impeded their widespread use in single-molecule studies. We used atomic force microscopic (AFM) imaging to demonstrate the 1:1 formation of UvrB-QD conjugates based on an antibodysandwich method. We show that an agarose gel-based electrophoresis mobility shift assay and AFM can be used to evaluate the DNA binding function of UvrB-QD conjugates. Importantly, we demonstrate that quantum dots can serve as a molecular marker to unambiguously identify the presence of a labeled protein in AFM images.In recent years, quantum dot (QD) bioconjugates have become increasingly popular in fluorescence experiments due to their narrow spectral emission width, strong emission intensity, small size, and good photostability. 1,2 Their strong emission intensities allow single quantum dots to be visualized by epifluorescence microscopy, while individual molecules of green fluorescent protein (GFP) and other synthetic fluorophores require the use of total internal reflection fluorescence (TIRF) microscopy to enhance the signal-tonoise ratio. 3 The unique properties of quantum dots also enable long-term tracking and monitoring of fast dynamics in single-molecule fluorescence microcopy studies. However, applications of protein-QD conjugates have so far been limited to antibodies for cell imaging, Western blot analysis, and fluorescence in situ hybridization. [4][5][6][7][8] Enzymatic studies of proteins using quantum dot conjugates have been limited to only a few proteins such as myosin, dynein, actin filaments, Rdh54, and Msh2-Msh6. 9-14 Several barriers are commonly encountered when using quantum dot labeled proteins in single-molecule studies. For example, conjugation of a quantum dot to a protein can potentially interfere with protein-protein and protein-ligand interactions. In addition, it is possible to conjugate more than one protein to a single functionalized quantum dot. Although gel electrophoresis in Supporting Information Available: Additional information includes Materials and Methods as well as figures showing more than one particles attached to a quantum dot at a 1:1 ratio of QD:UvrB-Ab, the aggregation of quantum dots in the presence of biotinylated HA antibody, assays confirming the nicking of the 517 base pair PCR fragment, statistical analysis of position distributions of UvrA and UvrB-QD on non-nicked DNA, and examples of UvrB-QD and UvrA-UvrB-QD conjugates on DNA. Atomic force microscopy (AFM) is a powerful single-molecule technique for studying biomolecular interactions. This technique can produce topographic images at high resolution (typically ≥10 nm). 16,17 However, for more complex, heteromeric assemblies, which are ubiquitous in many biological processes, using AFM imaging, we cannot always distinguish between different ...

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair

“…AFM-derived volumes have been used extensively in studies examining the oligomeric states of multicomponent complexes and to ascertain the nature of proteinprotein interactions of globular proteins (39,40). For our AFM studies, 517-bp PCR fragments were produced as the undamaged DNA substrate and the fragments were subjected to UV-irradiation to generate the damaged DNA (41,42). AFM analyses of UV-DDB in the presence of undamaged DNA (SI Appendix, Fig.…”

Single molecule techniques in DNA repair: A primer

“…AFM has proven invaluable in the study of DNA repair [45–49]. For example, UvrA and UvrAB, involved in NER, have been shown using AFM to preferentially bind to DNA ends on undamaged DNA substrates [45,46].…”

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

“…For protein–protein complexes, the volume is directly proportional to the protein molecular weight (Ratcliff & Erie, 2001; Yang et al, 2003), and this relationship also appears to hold reasonably well for most protein–DNA complexes. Volume analysis has been used to determine the stoichiometry for many repair proteins (e.g., see Chelico, Sacho, Erie, & Goodman, 2008; Wang et al, 2006; Wang, Tessmer, Croteau, Erie, & Van Houten, 2008; Xue et al, 2002). For protein–DNA complexes, the first peak in the volume distribution usually represents the volume of a single protein on the DNA.…”