Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers - PubMed (original) (raw)

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Ciro Cecconi et al. Eur Biophys J. 2008 Jul.

Abstract

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

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Figures

Fig. 1

Fig. 1

Attachment of DNA handles to protein molecules. a Schematic of the reactions used to: (1) activate protein’s cysteine residues with DTDP, and (2) attach DNA molecules to activated protein thiols. b Time course of the release of pyridine-2-thione during the activation of RNase H*Q4C/V155C (red dots), T4L*K16C/D159C (green triangles), and of the cysteine-free variant RNase H* (blue dots). Inset, activation of T4L*T21C/K124C with (purple dots) and without 3M GdmCl (orange triangles)

Fig. 2

Fig. 2

Attachment of 558 bp DNA handles to protein molecules. a–c Show atomic force microscopy images of DNA handles alone, RNase H bound to one handle and RNase H bound to two handles, respectively. d SDS-PAGE analysis of the sequential attachment of DNA handles to T4L*T21C/K124C. This 4% gel shows handles alone (lane 1, position a and c; the latter band is the result of the reaction between two DNA handles through their thiol groups), T4 lysozyme bound to one handle (lanes 2 and 5, positions b), and T4 lysozyme bound to two DNA handles (lane 5, position d). Lanes 3 and 4 show two different DNA ladders. As expected, the bands in position b and d are not present when the sample is treated with proteinase K (data not shown). When the sample is run on a native gel, the molecules electroeluted from band d all display the same structure of the two DNA-protein-DNA complexes shown in c (data not shown). The gel was stained with SYBR Green II and is shown in gray scale. e Time course of the release of pyridine-2-thione during the one-step attachment of both DNA handles to RNase H. The kinetics of the reaction is biphasic. f Four percent SDS-PAGE gel of handles alone (lane 1), and of the product of the onestep attachment (lane 2). The bands corresponding to RNase H bound to one or two handles are visible in position b and d, respectively

Fig. 3

Fig. 3

Synthesis of protein polymers and their attachment to DNA handles. a Schematic of the reaction used to polymerize proteins. b A 4–20% gradient SDS-PAGE gel of the polymerization reaction of RNase H stained with SYPRO Red and shown in gray scale. c A 4–20% gradient SDS-PAGE gel of poly-RNase H alone (lane 1), poly-RNase H after reaction with 40 bp DNA handles bearing a 5′-thiol group (lane 2), and poly-RNase H after reaction with 40 bp DNA handle (lane 3). The gel was stained both with SYBR Green II and SYPRO Red

Fig. 4

Fig. 4

Effect of DNA handles on protein stability. a Temperature melts of T4L*D61C/D159C alone (blue dots) and attached to two single stranded 20 base DNAs (light blue triangles). The data were normalized to show the fraction of protein unfolded at each temperature. In both cases, the two-state fits to the data yield a _T_m of 57°C. b CD spectra of T4L*D61C/D159C attached to two single stranded 20 base DNAs, before (red dots) and after (blue dots) thermal unfolding. The CD spectrum of the protein alone is shown in the inset (green dots)

Fig. 5

Fig. 5

Experimental set-up and force–extension curves. a Schematic of a single globular protein attached to polystyrene beads through DNA handles. One handle is derivatized with a 5′ biotin moiety, which interacts with a streptavidin-coated bead held in place at the end of a pipette by suction. The other handle is derivatized with a 5′ digoxigenin moiety, which interacts with an antibody-coated bead held in a laser trap. b Force–extension cycles obtained by stretching and relaxing a single RNase H molecule (see also Cecconi et al. 2005). c Extension versus time trace of an RNase H molecule held at constant force, F = 6.0 pN. d Schematic of a protein polymer attached to polystyrene beads through DNA handles. e Force–extension cycles obtained by stretching and relaxing a tetramer of RNase H molecules multiple times using long handles. f Force–extension cycles obtained by stretching and relaxing a polymer of RNase H molecules (likely a heptamer) multiple times using short handles

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