9 research outputs found
<sup>19</sup>F NMR as a Tool for Monitoring Individual Differentially Labeled Proteins in Complex Mixtures
The ability to monitor the behavior
of individual proteins in complex
mixtures has many potential uses, ranging from analysis of protein
interactions in highly concentrated solutions, modeling biological
fluids or the intracellular environment, to optimizing biopharmaceutical
co-formulations. Differential labeling NMR approaches, which traditionally
use <sup>15</sup>N or <sup>13</sup>C isotope incorporation during
recombinant expression, are not always practical in cases when endogenous
proteins are obtained from an organism, or where the expression system
does not allow for efficient labeling, especially for larger proteins.
This study proposes differential labeling of proteins by covalent
attachment of <sup>19</sup>F groups with distinct chemical shifts,
giving each protein a unique spectral signature which can be monitored
by <sup>19</sup>F NMR without signal overlap, even in complex mixtures,
and without any interfering signals from the buffer or other unlabeled
components. Parameters, such as signal intensities, translational
diffusion coefficients, and transverse relaxation rates, which report
on the behavior of individual proteins in the mixture, can be recorded
even for proteins as large as antibodies at a wide range of concentrations
NMR calculation statistics for an ensemble of the 20 lowest energy structures of ALYREF fragment (ALYREF<sup>54–155</sup>) bound to ORF57<sup>103–120</sup> (PDB code 2YKA).
<p>NMR calculation statistics for an ensemble of the 20 lowest energy structures of ALYREF fragment (ALYREF<sup>54–155</sup>) bound to ORF57<sup>103–120</sup> (PDB code 2YKA).</p
Typical effects of complex formation and RNA→protein ST on selected signals of ALYREF<sup>1–155</sup> and ORF57<sup>8–120</sup>.
<p>The <sup>1</sup>H dimension slices through <sup>1</sup>H-<sup>15</sup>N-correlation spectra are displayed for three representative signals of each protein, on the left for ALYREF and on the right for ORF57. The residue assignments in the free form are labeled at the top, and same signals are shown below each other for different complexes, as indicated. First type of signal (ALYREF<sup>M1</sup> and ORF57<sup>E24</sup>) is not significantly affected by any complex formation, or ST. Second type (ALYREF<sup>A34</sup> and ORF57<sup>Y81</sup>) is not affected much by protein and marginally affected by RNA binding, but is altered or displays significant ST effect in the ternary complex (percentage drop in signal intensity is indicated in blue, and ST spectral traces shown in red). These are residues likely contributing to cooperative ternary complex formation, forming contacts with RNA. Third type of signals originates from the structured regions of proteins (ALYREF<sup>A104</sup> and ORF57<sup>R111</sup>). ALYREF<sup>A104</sup> signal is not significantly affected in protein-protein complex, but shows significant increase in ST effect in the ternary complex, suggesting that ORF57 recruits RNA to the proximity of this residue. ORF57<sup>R111</sup> signal is broadened beyond detection in protein-protein complex, and remains broadened in the ternary complex. For this signal strong ST effect is observed when in complex with RNA. This residue is involved in initial viral RNA recognition, but then RNA is displaced from this site by ALYREF binding.</p
Overview of RNA interaction sites mapped on ORF57<sup>8–120</sup> and ALYREF<sup>1–155</sup> using different approaches.
<p>Backbone amide chemical shift change (δCS) and saturation transfer (ST) data are shown for ORF57<sup>8–120</sup> (A) and ALYREF<sup>1–155</sup> (B) with different ligands added as labeled. Crosses indicate residues with signal broadened beyond detection. For δCS, large (>0.1) and moderate (>0.04) chemical shift changes of each protein relative to signals in its free state, are shown as solid and broken bars, respectively. For ST data, signal intensity ratios significantly different from the background mean values are represented by solid (>6 SD) and broken (>3 SD) bars, respectively. Labels indicate the state of the protein for each dataset, data shown for interactions with non-specific RNA (green), ORF57 specific RNA (red/orange) and ALYREF-ORF57-RNA complex (blue).</p
Fluorescent studies and simulations of the ternary complex formation between ORF57, O, fragment of viral RNA, R, and ALYREF, A.
<p>(<i>A</i>) Simultaneous non-linear fit of normalized fluorescence-derived parameters <i>Δλ<sup>N</sup><sub>bcm</sub></i> (blue shift of emission signal) and <i>ΔI<sup>N</sup></i> (fluorescence quenching) to the non-redundant three-equation model using DynaFit software, to obtain <i>K<sub>d</sub></i> for the ternary complex. The experimental values of thus determined <i>K<sub>d</sub><sup>OA+R</sup></i>, as well as <i>K<sub>d</sub></i>'s for other complexes measured earlier, are summarized on two illustrative thermodynamic cycles for the ternary complex assembly presented on panels <i>(B)</i> and <i>(C)</i>. Simulations (using COPASI software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003907#ppat.1003907-Hoops1" target="_blank">[67]</a>) for these two possible cycles illustrate an increase in the concentration of ternary ORF57-RNA-ALYREF complex when ORF57 is added to 10 µM equimolar mixture of ALYREF and RNA, assuming the simplest four-state equilibrium model (<i>B</i>), or six-state model which additionally takes into account weak nonspecific ALYREF-RNA binding (<i>C</i>). The arrow marks a point where all the components of ternary complex are present in equimolar amounts. The presence of equimolar ORF57 significantly increases the concentration of RNA in complex with ALYREF (i.e., [OAR] vs [AR]<sub>[O] = 0</sub>). The baseline concentrations [AR]<sub>[O] = 0</sub> are indicated on the panel (<i>C</i>) on the left, assuming two different conservative estimates for values of <i>K</i><sub>d</sub> for nonspecific ALYREF-RNA binding.</p
Comparison of ALYREF<sup>54–155</sup>-ORF57<sup>103–120</sup> with ALYREF<sup>54–155</sup>-ICP27<sup>103–138</sup> and U2AF complex structures.
<p>(A) Overlay of the RRM domains of ALYREF in complex with ICP27<sup>103–138</sup> (green and magenta, PDB code 2kt5 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003907#ppat.1003907-Tunnicliffe1" target="_blank">[30]</a>) and ORF57-bound ALYREF determined here (cyan and orange), demonstrating the shift in α-helix 1 position. (B) ALYREF<sup>54–155</sup> in complex with ORF57<sup>103–120</sup>, determined here. (C) Backbone amide weighted chemical shift changes (δCS) in the ALYREF<sup>54–155</sup>-ORF57<sup>103–120</sup> complex are emphasized by color. δCS>0.3 are red, 0.15–0.299 orange, 0.05–0.149 yellow, prolines are blue and regions unaffected are green. (D) U2AF<sup>35</sup> in complex with U2AF<sup>65</sup> (PDB code 1jmt, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003907#ppat.1003907-Kielkopf1" target="_blank">[50]</a>). (E) ALYREF<sup>54–155</sup> in complex with ICP27<sup>103–138</sup>, previously determined (pdb 2kt5). (F) Backbone amide weighted chemical shift changes (δCS) in the ALYREF<sup>54–155</sup>-ICP27<sup>103–120</sup> complex with the same coloring as panel C.</p
Probing ORF57-RNA binding by mutations and UV cross-linking, and ALYREF-ORF57-RNA remodeling assay.
<p>(<i>A</i>) Purified hexa-histidine tagged GB1 (negative control) or ORF57<sup>8–120</sup> WT and point mutants (as labeled) were incubated with end-labeled 14merS RNA oligonucleotide before the mixture was subjected to UV cross-link (+) or not (−). Similarly in the remodeling assay, WT ORF57<sup>8–120</sup> was incubated with end-labeled 7merS (<i>B</i>) or 14merS (<i>C</i>), before the mixture was added to purified GST-ALYREF immobilized onto glutathione coated beads. Purified complexes were eluted in native conditions and UV cross-linked (+) or not (−). All samples were finally analyzed on 15% SDS-PAGE stained with Coomassie blue and by PhosphoImaging.</p
Model of the passage of RNA between ORF57 and ALYREF.
<p>Local protein interactions with RNA are detected by moderate (orange) and large (red) saturation transfer effects (represented by ‘lightning bolts’) observed by ST-HSQC or ST-IDIS-TROSY and mapped onto respective regions. Broadened residues are colored light-yellow. Linked black circles represent a position for transiently bound RNA. ALYREF (green) binds RNA 14merS weakly via its RRM and N-terminal regions (A), whereas ORF57 (blue) binds 14merS tightly mainly via the R-b helix and also the aa81–92 region (B). Interaction of ORF57-RNA complex with ALYREF partially displaces the RNA from the R-b helix, while RNA maintains contact with ORF57 aa81–92 and also forms new contacts with ALYREF's aa22–48 and helix-2 of the RRM domain (C). The RNA contacts with ALYREF within the ALYREF-ORF57-RNA ternary complex are more abundant than for just ALYREF-RNA, and thus ORF57 enhances the interaction of viral RNA with ALYREF.</p
Investigating Liquid–Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl
Liquid–liquid
phase separation (LLPS) of monoclonal antibody
(mAb) formulations involves spontaneous separation into dense (protein-rich)
and diluted (protein-lean) phases and should be avoided in the final
drug product. Understanding the factors leading to LLPS and ways to
predict and prevent it would therefore be highly beneficial. Here
we describe the link between LLPS behavior of an IgG1 mAb (mAb5),
its solubility, and parameters extracted using <sup>1</sup>H NMR spectroscopy,
for various formulations. We show that the formulations demonstrating
least LLPS lead to the largest mAb5 NMR signal intensities. In the
formulations exhibiting the highest propensity to phase-separate the
mAb NMR signal intensities are the lowest, even at higher temperatures
without visible phase separation, suggesting a high degree of self-association
prior to distinct phase separation. Addition of arginine glutamate
prevented LLPS and led to a significant increase in the observed mAb
signal intensity, whereas the effect of arginine hydrochloride was
only marginal. Solution NMR spectroscopy was further used to characterize
the protein-lean and protein-rich phases separately and demonstrated
that protein self-association in the protein-rich phase can be significantly
reduced by arginine glutamate. Solution NMR spectroscopy may be useful
as a tool to assess the propensity of mAb solutions to phase-separate