Conformational Change in Unsolvated Trp-cage Protein Probed by Fluorescence

We report the first direct measurements of the unfolding of a protein, Trp-cage, in the gas phase using laser-induced fluorescence of protein ions in a heated quadrupole ion trap. The changes in enthalpy and entropy associated with the observed conformational change are obtained by fitting a two-state model of protein unfolding to the fluorescence intensities plotted versus temperature. The enthalpy and entropy changes for the 2+ and 3+ charge states are greater than the values measured in solution and depend on charge state

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosyl­methionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosyl­methionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes

Quantitative Proteomic Approach for Cellulose Degradation by <i>Neurospora crassa</i>

Conversion of plant biomass to soluble sugars is the primary bottleneck associated with production of economically viable cellulosic fuels and chemicals. To better understand the biochemical route that filamentous fungi use to degrade plant biomass, we have taken a quantitative proteomics approach to characterizing the secretome of Neurospora crassa during growth on microcrystalline cellulose. Thirteen proteins were quantified in the N. crassa secretome using a combination of Absolute Quantification (AQUA) and Absolute SILAC to verify protein concentrations. Four of these enzymes including 2 cellobiohydrolases (CBH-1 and GH6-2), an endoglucanase (GH5-1), and a β-glucosidase (GH3-4) were then chosen to reconstitute a defined cellulase mixture in vitro. These enzymes were assayed alone and in mixtures and the activity of the reconstituted set was then compared to the crude mixture of N. crassa secretome proteins. Results show that while these 4 proteins represent 63–65% of the total secretome by weight, they account for just 43% of the total activity on microcrystalline cellulose after 24 h of hydrolysis. This result and quantitative proteomic data on other less abundant proteins secreted by Neurospora suggest that proteins other than canonical fungal cellulases may play an important role in cellulose degradation by fungi

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosyl­methionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes

Rv2131c from <i>Mycobacterium tuberculosis</i> Is a CysQ 3′-Phosphoadenosine-5′-phosphatase

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3′-phosphoadenosine-5′-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3′-phosphoadenosine 5′-phosphate (PAP) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the <i>Mtb</i> genome suggests the presence of a CysQ homologue encoded by the gene <i>Rv2131c</i>. However, a recent biochemical study assigned the protein’s function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed <i>Rv2131c</i> heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl₂. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a <i>K</i>_m (8.1 ± 3.1 μM) and <i>k</i>_cat (5.4 ± 1.1 s⁻¹) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for <i>myo</i>-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the <i>Rv2131c</i>-encoded enzyme to dephosphorylate PAP and PAPS <i>in vivo</i> was confirmed by functional complementation of an <i>Escherichia coli</i> Δ<i>cysQ</i> mutant. Taken together, these studies indicate that <i>Rv2131c</i> encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism

TthCsm-mediated ssDNA cleavage is sequence-independent and endonucleolytic.

<p><b>(A)</b> TthCsm-mediated cleavage of a 5′-³²P-radiolabeled complementary or noncomplementary dsDNA (dsDNA, C or NC), ssDNA (C or NC), or a duplex with a 40-nt long mismatch in the center (bubble DNA) was tested in the presence of complementary target ssRNA, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170552#pone.0170552.g005" target="_blank">Fig 5B</a>. <b>(B)</b> TthCsm was incubated with 5′-³²P-radiolabeled 25 nt-long ssDNA substrates with different sequences in the presence of a complementary target ssRNA and MnCl₂. Sequences included all four nucleotides (all nt), all except adenines (-A), thymines (-T), guanines (-G), or cytosines (-C). The reaction was carried out for 60 minutes, either with (+) or without (-) TthCsm added.</p

Reconstitution and RNA cleavage activity of a <i>T</i>. <i>thermophilus</i> Csm complex (TthCsm) purified from <i>E</i>. <i>coli</i> with a defined crRNA species.

<p><b>(A)</b> Components of the CRISPR locus and effector complexes of the <i>T</i>. <i>thermophilus</i> Type III-A Csm system. The complex is shown with 5 copies of Csm3 and 4 copies of Csm2, but complexes with different numbers of these two subunits also exist. The CRISPR-4 locus associated with the system is shown (repeat is designated by R and spacer by S). The spacer 4.5 used for complex reconstitution encodes for one of the most abundant crRNAs found in the host organism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170552#pone.0170552.ref021" target="_blank">21</a>]. (<b>B</b>) Reconstitution and purification of TthCsm in <i>E</i>. <i>coli</i>. A plasmid containing genes encoding for Cas10/Csm1, and Csm2-5, with a His₁₀ tag on Csm5, was co-transformed into <i>E</i>. <i>coli</i> with a plasmid containing genes for expression of <i>T</i>. <i>thermophilus</i> Cas6A and a single CRISPR array containing one copy of spacer 4.5. The purification steps are indicated. (<b>C</b>) TthCsm was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis following purification. Csm subunits are labeled, and a molecular weight ladder (M) is in the left lane (masses are given in kilodaltons). A GroEL contaminant (asterisk) was also identified by mass spectrometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170552#pone.0170552.s006" target="_blank">S2 Table</a>). (<b>D</b>) TthCsm-mediated cleavage of a complementary (C) or noncomplementary (NC) ³²P-labeled ssRNA oligonucleotide was tested in the presence of 2 mM MgCl₂. Samples taken at 0, 5, 30, and 60 minutes after TthCsm addition were analyzed by denaturing PAGE. (<b>E</b>) Schematic of crRNA processing in Type III CRISPR-Cas systems is shown on the left. Pre-crRNAs are cleaved by Cas6 to generate an intermediate, which is then trimmed at the 3’-end, resulting in mature crRNAs. On the right, nucleic acids associated with the Csm complex were extracted and analyzed by denaturing PAGE. An ssDNA oligonucleotide ladder (M) was loaded in the right-most lane and nucleotide lengths are indicated.</p

TthCsm specifically recognizes and binds ssRNA through complementarity to its crRNA.

(A) An increasing concentration of TthCsm, from 0–300 nM, was incubated with 0.5 nM 32P-labeled ssRNA target that was complementary (C) or noncomplementary (NC) to the crRNA guide sequence, and analyzed by an EMSA. (B) 100 nM TthCsm was incubated with 0.5 nM 32P-labeled target ssRNA and increasing concentrations of unlabeled complementary ssRNA (ssRNA, C), noncomplementary ssRNA (ssRNA, NC), complementary ssDNA (ssDNA, C), or noncomplementary ssDNA (ssDNA, NC) competitor (0–1 μM). Samples were assayed for binding of the probe using an EMSA, as in (A).</p

Metallothionein-Cross-Linked Hydrogels for the Selective Removal of Heavy Metals from Water

Metallothionein-Cross-Linked Hydrogels for the Selective Removal of Heavy Metals from Wate