Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Magnesium-induced folding of SL ribozyme constructs. Peak E shifts from the Gaussian fits of the FRET histograms at Mg2+ concentrations from 0.01 mM to 1 M for the SL (▪) and WT (⋄) 2WJ, 3WJ, and 4WJ ribozymes (parts A, B, and C, respectively). Peak E values for a control donor-acceptor labeled DNA molecule with dyes separated by 14 base pairs are shown for comparison as solid triangles in the 3WJ graph in part B.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Control, Labeling, Comparison

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Ratiometric spFRET data for the WT 4WJ ribozyme. (A) Example of donor (black) and acceptor (gray) emission bursts observed in spFRET experiments. A time trace is presented for the WT 4WJ ribozyme at a concentration of 100 pM in 50 mM Tris/HCl buffer with 0.1 mM Mg2+ at room temperature, and the data integration time per point was 0.5 ms. Fluorescent donor and acceptor bursts are observed above background. A buffer (background) time trace is also shown for comparison. Only bursts above a threshold of 40 counts (sum of donor and acceptor signals) were used to construct FRET efficiency histograms. (B) FRET efficiency histogram derived from calculation of FRET efficiencies from each accepted donor/acceptor event above a threshold of 40 counts, showing the docked and undocked subpopulations (see text), as well as the zero-E peak. The solid lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Concentration Assay, Comparison, Construct, Derivative Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: FRET efficiency histogram for a G11I mutant of the 4WJ ribozyme at 0.1 mM Mg2+, using the same conditions as for data presented in Fig. 3. Solid lines show fits using Gaussian functions. The relative area of the high efficiency (docked) peak is markedly reduced relative to the wild-type 4WJ ribozyme (Fig. 3), reflecting a destabilized tertiary structure for the mutant.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Mutagenesis

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Schematic representation of WT ribozyme constructs used for spFRET studies (upper panel). FRET efficiency histograms (bars) for wild-type 2WJ, 3WJ, and 4WJ hairpin ribozymes, showing undocked and docked ribozyme subpopulations at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Single-loop 2WJ, 3WJ, and 4WJ ribozyme constructs (upper panel) and corresponding FRET efficiency histograms at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Kinetic information from peak shape analysis of spFRET histograms. (A) Overlay of FRET efficiency histograms for SL (bold solid line) and WT 4WJ (data as bar graph, Gaussian fit as solid line) ribozymes at 10 mM Mg2+, emphasizing the asymmetric peak shape for the SL 4WJ. The error bars represent single standard deviations. (B) Overlay of FRET histograms from two-state simulations (lines) and experimental data (bars; errors represent single standard deviations) for the SL 4WJ at 10 mM Mg2+. The extended-undocked (EU)–quasi-docked (QD) equilibrium scheme is shown in the inset. Simulated histograms are shown for forward/reverse (k1/k−1) rate constants of 20 × 103/6.8 × 103 s−1 (bold solid line, 1), 41 × 103/14 × 103 s−1 (bold dashed line, 2), and 10 × 103/3.4 × 103 s−1 (bold dash-dot-dashed line, 3) with a corresponding equilibrium constant of 3 in favor of the quasi-docked state in all cases. See text for details.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Using AFM to detect the presence, absence, or the blocking of isopeptide bonds in the Spy0128 construct. (A) The native intramolecular isopeptide bond in Spy0128 prevents the protein from unfolding and extending under force. Unfolding sawtooth pattern obtained by stretching the unmodified Spy0128 construct. These traces only show the three I91 fingerprints with short initial extensions (Li, defined as the extension to the first I91 unfolding) and contour length increments of 29 nm, measured using fits of the worm-like chain model of polymer elasticity (solid lines). Data are from ref. 24. (B) Mutant Spy0128 (E258A) where the isopeptide bond is not formed. In this case, the sawtooth patterns show two additional high-force unfolding events that always follow the I91 fingerprints and have large contour length increments of ∼50 nm. Data from ref. 24. (C) Spy0128 constructs modified by the isopeptide blocker show a very different type of sawtooth pattern, with long initial extensions marked by a series of random low-force peaks observed always before the I91 fingerprint. (D) Histogram of initial extensions (Li) measured from sawtooth patterns obtained from Spy0128 constructs that were coexpressed with the isopeptide blocker (n = 698). A Gaussian fit (solid line) marks three distinct peaks at 41, 80, and 130 nm. The peak at 41 nm corresponds to unmodified Spy0128 constructs. The peaks at 80 and 130 nm closely correspond to the extensions predicted if one or both isopeptide bonds were blocked and the modified protein was unable to fold.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Blocking Assay, Construct, Polymer, Mutagenesis, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Mechanical properties of HaloTag-purified Spy0128 constructs. Using Promega paramagnetic beads, we enrich the fraction of proteins that have been modified by the blocking peptide. The proteins are subsequently detached from the beads using the TEV protease. From these purified proteins we obtained three types of sawtooth patterns: (A) Unmodified Spy0128 constructs with short initial extensions (Li < 50 nm) and (B) showing a single modification (Li ∼ 50–100 nm) or (C) two modifications (Li > 100 nm). (D) Histogram of initial extensions (n = 453). A Gaussian fit identifies three peaks at 34, 86, and 135 nm. (E) Two-dimensional histogram showing the density of unfolding forces (FU) versus the contour length increments (∆Lc) for the isopeptide-blocked Spy0128. Modified pilin proteins unfold at forces below 100 pN and do not show any well-defined unfolding pathway. Remarkably, 14% of the traces (n = 40) show long initial extensions without any unfolding event, suggesting that the modified Spy0128 extends as an unstructured polymer. (F) Mutant Spy0128 E258A unfolds through well-defined unfolding pathways with high mechanical stability, either through a single event with FU ∼ 300 pN or through an intermediate with FU1 ∼ 180 pN and FU2 ∼ 110 pN. Data from ref. 24.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Purification, Construct, Modification, Blocking Assay, Polymer, Mutagenesis