GEF activity of purified Ric-8A fragments defined by limited trypsinolysis and secondary structure analysis.

<p>(A) Coomassie-stained SDS PAGE analysis of Ric-8A after trypsinization for the times indicated below each lane; unique fragments are identified by colored asterisks. (B) Electrospray mass spectrometric analysis of Ric-8A tryptic digest fragments extracted from the SDS PAGE gel shown in panel A; peaks identified by asterisks refer to corresponding bands shown in panel A. Fragment masses (Da) are indicated at each peak position. (C) Amino acid sequence of rat Ric-8A; cylinders indicate helical segments predicted using JPRED <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Cole1" target="_blank">[51]</a>. Residue codes colored red indicate sites of proteolytic cleavage (see panel A). Residue codes in green indicate N or C-termini of recombinant Ric-8A fragments engineered to coincide approximately with proteolytic sites or predicted secondary structure boundaries: ΔC492 denotes the Ric-8A fragment comprising residues 1–492. Both N-terminal truncations ΔN12 and ΔN38 were also C-terminally truncated at residue 492 and comprised residues 12–492 and 38–492, respectively. (D) Kinetics of intrinsic (open symbols) or Ric-8A-stimulated (filled symbols) GDP release (squares) from, or GTPγ binding to (circles) myristoylated Gαi1 were determined by a filter binding assay using radiolabeled nucleotides as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Tall1" target="_blank">[4]</a>. Upper left panel, Gαi1 (200 nM) nucleotide binding and release in the presence of full-length Ric-8A (200 nM); lower left panel, ΔC492Ric-8A (200 nM); upper right panel, ΔC453Ric-8A (200 nM); lower right panel, Gαi1 alone. Data for each panel are normalized to maximum GDP released or GTPγS bound in a single experiment. Data points represent the average of three experiments; standard deviation from the mean is <10%. Time course of GTPγS binding in the absence of Ric-8A, shown at lower right, is replicated in the other panels for comparison. (E), Histogram showing relative rates of Gαi1 GDP release (red bars) and GTPγS binding (blue bars) catalyzed by Ric-8A and Ric-8A truncation mutants (200 nM). Error bars represent +/− one standard deviation of the apparent first-order rate constants determined in three replicates.</p

Nucleotide-free Gαi1 is relatively unstructured in comparison to Gαi1•GDP, but regains helical secondary structure in the complex with Ric-8A.

<p>Circular dichroic spectra were normalized as mean residue elipticity, and predicted secondary structure assignments are: Ric-8A, <i>red</i>: 87% α-helix; Ric-8A:Gαi1[ ], <i>black</i>: 87% α-helix, 0.5% β-strand; Gαi1•GDP, <i>green</i>: 51% α-helix, and 11% β-strand; Gαi1[ ], <i>blue</i>, 38% α-helix, 9% β-strand.</p

Intrinsic and Ric-8A-catalyzed GTPγS binding rates of the Gαi1 proteins used in this study.

<p>Intrinsic and Ric-8A-catalyzed kinetics of binding of GTPγS to wild-type Gαi1, W258A-Gαi1, NΔ25Gαi1, GαiCΔ9 and Gαi1-GαsC12 were measured using a fluorescence binding assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Thomas1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Higashijima1" target="_blank">[47]</a>. 400 µl of protein (1 µM) in the GDP bound form was equilibrated for 10–15 min at 25°C in a cuvette. A 10-fold excess of GTPγS was added and fluorescence at 340 nm upon excitation at 290 nm was monitored in the absence (open bars) or presence (filled bars) of Ric-8A (1 µM). Error bars represent +/− one standard deviation apparent first-order rate constants determined in three replicates.</p

¹H-¹⁵N TROSY-HSQC spectrum of Gαi1 shows extensive peak broadening and intensity loss upon binding of Ric-8A.

<p>(A) ¹H-¹⁵N HSQC-TROSY spectra were acquired for [¹⁵N]Gαi1•GDP, and (B) Ric8A:[¹⁵N]Gαi1[ ]. (C) After acquisition of the Ric-8A:[¹⁵N]Gαi1[ ] spectrum, a five molar excess of GTPγS was added to induce dissociation of Ric-8A and formation of Gαi1•GTPγS. After a short incubation, the free Ric-8A was removed by adsorption to IMAC resin, and the ¹H-¹⁵N TROSY-HSQC spectrum of the sample was recorded. Protein concentration, acquisition and processing parameters and contour levels in all panels are the same.</p

Nucleotide-free Gαi1 bound to Ric-8A exhibits rapid hydrogen/deuterium exchange kinetics relative to the Gαi1•GDP complex.

<p>(A) Mass distribution for Gαi1•GDP measured at fixed time points (see panel C) after dilution into D₂O; the mass distribution at the zero time point, before exchange was initiated, corresponds to the red peak centered at 40.1 kDa. The average of the Gαi1 mass distribution increases as H/D exchange reaction proceeds. (B) Mass distribution for Gαi1[ ] in complex with Ric-8A measured at fixed time points after dilution into D₂O; note that the Gαi1[ ] mass distribution becomes multimodal as the H/D exchange reaction proceeds. (C) The increase in mass (Da), determined at the centroid of the mass distribution of Gαi1•GDP (black squares) and Gαi1[ ] derived from the complex with Ric-8A (red circles) is plotted as a function of time after rapid dilution from aqueous buffer into D₂O.</p

Ric-8A provides limited protection of nucleotide-free Gαi1 from trypsin digestion.

<p>Samples were incubated with TPCK treated trypsin at a 1∶1000 molar ratio (trypsin∶sample) at 4°C, withdrawn at the indicated time points, separated by SDS-PAGE and visualized by Coomassie blue staining. (A) Gαi1•GDP: lanes from left to right: molecular weight markers, M; untreated Gαi1•GDP, U; samples digested for 10 and 25 minutes. (B) nucleotide-free Gαi1[ ]: markers, M; untreated Gαi1[ ], U; samples digested for 5 and 10 minutes. Mass spectroscopic analysis identifies band 1 as Gαi1 residues 21–179: observed/calculated mass 17,761/17,774 Da. (C) Ric-8A: markers, M; untreated, U. (D) Ric-8A: after 5, 10 and 15 minutes of trypsin digestion. Mass analysis identifies band 1 as Ric-8A residues 1–408: 46,218/46,207 Da; band 2, residues 72–378: 34,815/34,799; band 3, residues 141–348: 23,834/23,804 Da; band 4, residues 62–178: 13,563/13,523 Da. (E) Ric-8A: Gαi1[ ] complex: markers, M; untreated Ric-8A:Gαi1[ ] complex; R and G indicate bands for intact Ric-8A and Gαi1, respectively; Ric-8A:Gαi1[ ] complex digested for 10 and 25 minutes. Mass analysis identifies band 1 as Ric-8A residues 141–348: 23,834/23,804 Da (present also as band 3 in Panel D); band 2, Gαi1 residues 17–191: 19,646/19,652 Da; band 3, Gαi1 residues 21–179: 17,753/17,761 Da (present as band 1 in panel B); band 4: Gαi1 residues 10–141: 14,532/14,520 Da.</p

Thermal denaturation properties of Gαi1 and Ric-8A are affected by their mutual interaction.

<p>Temperature-dependence of heat capacity was measured by differential scanning calorimetry. Buffer baseline-corrected thermograms were recorded for Ric-8A (black trace), Gαi1[ ] (dashed blue trace), Gαi1•GDP (blue trace) and Ric-8A:Gαi1[ ] (green trace). The weighted average of the thermograms for Ric-8A and Gαi1[ ] (red dashed line) overlaps that of Gαi1[ ] in the temperature range below ∼37°C and that of Ric-8A above that temperature, and is distinct from the thermogram of Ric-8A:Gαi1[ ]. The inset shows a magnified view of the four thermograms and the weighted average function in the 20°C–45°C range.</p

Mechanism of N₂ Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H₂

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N₂ reduction relative to H₂ evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe–H–Fe hydrides on the active site FeMo-cofactor to yield H₂ is a key feature in the N₂ reduction mechanism. The N₂ reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N₂ reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N₂ reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N₂ reduction (1 atm) of 181 ± 5 nmol NH₃ min^–1 mg^–1 FeFe protein, for proton reduction (in the absence of N₂) of 1085 ± 41 nmol H₂ min^–1 mg^–1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C₂H₄ min^–1 mg^–1 FeFe protein. Under turnover conditions, N₂ reduction is inhibited by H₂ and the enzyme catalyzes the formation of HD when presented with N₂ and D₂. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N₂ reduction occurring by reductive elimination of H₂

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In <i>Escherichia coli</i>, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions <i>in vivo</i> and <i>in vitro.</i> Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In <i>Escherichia coli</i>, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions <i>in vivo</i> and <i>in vitro.</i> Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine