Internal dynamics of the 3-Pyrroline-N-Oxide ring in spin-labeled proteins

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy

Water Network in the Binding Pocket of Fluorinated BPTI–Trypsin ComplexesInsights from Simulation and Experiment

Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly

ATP binding status of recombinant CF IA factors.

<p>ATP binding status of recombinant CF IA factors.</p

Water Network in the Binding Pocket of Fluorinated BPTI–Trypsin ComplexesInsights from Simulation and Experiment

Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly

Gene expression in strains expressing Clp1 proteins carrying a mutant P-loop.

<p>(A) Northern analysis of total RNAs obtained from wild-type and <i>CLP1</i> D161A and <i>CLP1</i> K136A T137A mutant strains following growth in YPD. Probes were as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029139#pone-0029139-g001" target="_blank">figure 1C</a>. (B) qRT-PCR analysis of candidate mRNAs that were identified during gene expression profiling of <i>CLP1</i> D161A and <i>CLP1</i> K136A T137A mutant strains. Transcript levels are presented relative to wild-type <i>CLP1</i>, which was fixed at 100%. Data shown are the mean of three independent biological replicates and error bars indicate standard deviation.</p

Water Network in the Binding Pocket of Fluorinated BPTI–Trypsin ComplexesInsights from Simulation and Experiment

Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly

Coupled <i>in vitro</i> transcription/3′ end processing.

<p>(A) Western blot analysis of CF IA factors partially purified from strains expressing ProtA-Clp1 and ProtA-Clp1 K136A T137A, respectively. Factor purification included a high salt wash (1 M KCl) of bound material to probe stability and integrity of the protein complexes. Decreasing amounts of CF IA associated with ProtA-Clp1 or ProtA-Clp1 K136A T137A were analyzed for the presence of the four CF IA subunits Rna15, Pcf11, Clp1 and Rna14 as indicated on the right. (B) Schematic presentation of the transcription template that was used in transcription/3′ end processing reactions <i>in vitro</i>. The construct includes Gal4 binding sites, a <i>CYC1</i> promoter and five G-less cassettes; the length of the cassettes in nucleotides is indicated. The <i>CYC1</i> terminator has been inserted between the 100 and 120 cassettes; also indicated are specific sequence elements (EE: efficiency element; PE: positioning element; UUE: upstream U-rich element; DUE: downstream U-rich element; and the poly(A) site). <i>In vitro</i> transcription produces a 0.5 kb polyadenylated RNA and read-through transcription produces a RNA of more than 1.3 kb length. (C) Western analysis of ProtA-Rna15 expressing whole cell extracts before and after two consecutive rounds of depletion on IgG-agarose. Increasing amounts (1, 2 and 4 µl) of extract (XT) and of depleted extract (2× depl) were resolved by SDS-PAGE and following transfer on PVDF membrane ProtA-Rna15 was detected using an anti-HA-HRP secondary antibody that readily bound to the ProteinA moiety of the fusion protein. Ponceau S staining of a section of the membrane is shown to demonstrate comparable loading between extract and depleted extract. (D) <i>In vitro</i> transcription/3′ end processing reactions with CF IA depleted extracts in with or without adding back ProtA-Clp1 and ProtA-Clp1 K136A T137A purified CF IA factors as indicated. On the top of each panel is indicated the salt concentration (mM KCl) applied in the final wash step of the protocol that was applied to purify CF IA factors associated with ProtA-tagged wild-type and mutant Clp1. G-less transcripts were separated on 8.3 M Urea 6% polyacrylamide gels. Migration of the G-less transcripts is indicated on the left. Gels at the bottom of each panel show the analysis of pre-mRNA 3′ end cleavage. Non-radioactive <i>in vitro</i> transcription reactions were performed and obtained RNAs were subjected to a ligation-mediated RT-PCR procedure. Formation of PCR product correlates with 3′ end cleavage activity and encompasses the site of poly(A) addition. (E) Quantification of the gels shown in (D). The obtained signals were normalized to uridine-content of the cassettes and presented as percentage of G-less transcription relative to the 100 cassette that is placed immediately upstream of the <i>CYC1</i> terminator; the site of poly(A) addition has been fixed at “0” bp. Termination and 3′ end formation is reflected by strength of transcription downstream of the terminator, and is indicated by 120/100, 131/100 and 145/100 signal ratios.</p

Effects of Clp1 depletion on gene expression.

<p>(A) Schematic presentation of the Clp1 fusion gene, which is expressed under the control of <i>GAL10</i> promoter. Encoded are an amino-terminal ubiquitin moiety fused in frame to Clp1 together with an HA-tag. Removal of the ubiquitin moiety by cellular deubiquitylases results in a protein with an arginine (R) at the amino-terminus creating an unstable protein that is targeted for degradation by the proteasome. A strain carrying this fusion gene is viable on YPGal medium but not on YPD, which contains repressive glucose as sole carbon source. (B) Western Blot of total extracts obtained from wild-type and <i>GAL-UBI-R-HA-CLP1</i> expressing strains following growth in YPD for the indicated times. Western blots were decorated with antibody against the HA-tag. Ponceau S staining served as control for equal loading. (C) Northern analysis of total RNAs obtained from wild-type and <i>GAL-UBI-R-HA-CLP1</i> expressing strains following growth in YPD for the indicated times. RNAs were detected with random prime labeled probes directed against the open reading frames as indicated on the left of each panel. The asterisk in the first panel marks a likely degradation product of the <i>UBI-R-HA-CLP1</i> mRNA. In <i>CYH2</i> and <i>NRD1</i> panels ‘ext’ denotes 3′ extended transcripts. 18S rRNA was detected with an end-labeled oligonucleotide and was used as control for equal loading. Schematically depicted on the right are features of analyzed and neighbouring genes with arrows indicating sites of 3′ end cleavage and polyadenylation.</p

Tracking Transient Conformational States of T4 Lysozyme at Room Temperature Combining X‑ray Crystallography and Site-Directed Spin Labeling

Proteins are dynamic molecules that can transiently adopt different conformational states. As the function of the system often depends critically on its conformational state a rigorous understanding of the correlation between structure, energetics and dynamics of the different accessible states is crucial. The biophysical characterization of such processes is, however, challenging as the excited states are often only marginally populated. We show that a combination of X-ray crystallography performed at 100 K as well as at room temperature and EPR spectroscopy on a spin-labeled single crystal allows to correlate the structures of the ground state and a thermally excited state with their thermodynamics using the variant 118R1 of T4 lysozyme as an example. In addition, it is shown that the surrounding solvent can significantly alter the energetic as well as the entropic contribution to the Gibbs free energy without major impact on the structure of both states

Internal Dynamics of the 3‑Pyrroline‑<i>N</i>‑Oxide Ring in Spin-Labeled Proteins

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy