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