Biocompatible Surfaces for Specific Tethering of Individual Protein Molecules

Abstract

We have characterized the biocompatibility of glass surfaces that were modified by protein layers or linear poly(ethylene glycol) (PEG) brushes with the aim of determining the optimal treatment for the immobilization of single biomolecules by specific attachment, which was done here using a streptavidin-biotin linkage. We have investigated the surface homogeneity by atomic force microscopy and the resistance to nonspecific binding by single-molecule detection of fluorescently labeled RNase H in a scanning confocal fluorescence microscope. The resistance to nonspecific binding of the surfaces was simply examined by omitting the biotinstreptavidin linkage in the surface preparation. We have also carried out fluorescence resonance energy transfer experiments to examine the ability of the surfaces to preserve the native state of specifically attached proteins via streptavidin-biotin linkage. Chemisorption and crosslinking of BSA or streptavidin to amino-functionalized glass yields excellent surface homogeneity and reproducibility, much better than physisorption onto bare glass. PEG 5000 films form the smoothest films and are very resistant to nonspecific protein adsorption, but PEG 2000 has significantly worse properties. Unlike protein-coated surfaces, PEG 5000 surfaces cause partial denaturation of the RNase H molecules, which we explain by intermingling of the PEG and polypeptide chains or by interaction of the underlying aminosilane layer with the protein. These results highlight the importance of a thorough surface characterization and design in fluorescence studies of immobilized single biomolecules.

Evidence of a Folding Intermediate in RNase H from Single‐Molecule FRET Experiments

“…Protein expression and purification were performed as previously described. [16,18,53] After initial acceptor labeling, singly labeled proteins were separated from unlabeled and doubly labeled fractions as well as free dye via HPLC (¾kta 900, GE Healthcare Europe, Freiburg, Germany) by using a cation exchange column (Resource S, GE Healthcare Europe). The procedure was repeated for subsequent donor labeling.…”

“…[6,[11][12][13][14][15][16] Most smFRET experiments have so far been carried out on two-state folders, that is, proteins that exhibit two-state behavior involving a folded (F) and an unfolded (U) state, owing to a clean separation of timescales of fluctuations between conformations within and transitions between the two states. [6,17] In previous smFRET studies, we investigated the structure and dynamics of surface-immobilized RNase H in the presence of the chemical denaturant GdmCl.…”

A practical guide to single-molecule FRET

“…Adhesion of nucleic acids to the PEG surface at pH < 7.0 can be reduced by further passivating the surface with sulfodisuccinimidyltartrate 46 . Strong interaction between denatured protein and linear PEG can be eliminated using branched PEG instead and they may serve as better passivating agents 47,48 . Histidine-tagged proteins can also be attached to the surface using chelated Ni 2+ or Cu 2+ groups 49 however, optimal orientational positioning and elimination of surface artifacts might sometimes require a spacer between the histidinetag sequence and the protein (Fig.…”

Direct Visualization of Single-Molecule DNA-Binding Proteins Along DNA to Understand DNA-Protein Interactions

“…An increase in the chain length of PEG to construct a defined tethered chain layer results in a decrease in the density of the PEG chain due to the exclusion volume effect. PEG with a molecular weight of 5,000 appears to find a compromise between the trade-offs to maximize the suppression of non-specific adsorption of protein molecules on a glass surface (Heyes et al, 2004;Malmstena et al, 1998;McNamee et al, 2007;Pasche et al, 2003;Yang et al, 1999). A general approach to the immobilization of PEG onto a glass surface involves coupling of PEG to amine groups that have been conjugated on the surface by silanization (Figure 1a).…”