24 research outputs found
Percentage of functional NTS1.
<p>The total protein content of the NiE, NTFT and NiE fractions was determined by the Amido Black method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>). The amount of total NTS1 in the NiE, NTFT and NiE fractions was determined by ImageJ analysis of Coomassie-stained gels. The number of functional NTS1 in pmoles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>) was converted into a milligram value using an NTS1 molecular mass of 96.5 kDa. The percent functionality was calculated as [(mg NTS1 by ligand binding analysis)/(mg NTS1 by SDS-PAGE analysis)]*100.</p
Stability of NTS1 in NT70 buffer with and without agonist.
<p>Receptors were incubated with [<sup>3</sup>H]NT (open squares) or kept in NT70 buffer without agonist (filled squares) at 4°C. [<sup>3</sup>H]NT binding was determined at the indicated time points. The data shown are from 2 independent experiments.</p
Stability of NTS1 in the absence of agonist.
<p>[<sup>3</sup>H]NT binding to the NTS1 fusion protein was recorded over time and half-lives were calculated. The amount of functional NTS1 remaining after 6 hrs (NiE and diluted NiE) and after 24 hrs (SN) was estimated from one phase exponential decay fits. Abbreviations: SN, supernatant after ultracentrifugation; NiE, Ni-NTA column eluate.</p
Summary of NTS1 purification.
<p>The percent values refer to the respective protein species in the NiE.</p
Functional and misfolded NTS1 during purification.
<p>Three different ways (columns A–C) were used to estimate the amounts of functional NTS1, misfolded NTS1 and contaminants in the NiE, NTFT and NTE fractions. All values in columns A–D are given in mg quantities. Abbreviations: NiE, Ni-NTA column eluate; NTFT, flow-through of NT column; NTE, NT column eluate; NA, not applicable.</p>1<p>The amount of functional NTS1 in the NiE, NTFT and NTE fractions was determined by [<sup>3</sup>H]NT binding (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Tables 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">4</a>). For example, the NiE contained 3678 pmoles or 0.355 mg functional NTS1.</p>2<p>The total amount of NTS1 protein in the NiE, NTFT and NTE fractions was determined by ImageJ analysis of Coomassie-stained gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>).</p>3<p>The amount of functional NTS1 in the NiE and NTE fractions was derived from the respective specific [<sup>3</sup>H]NT binding values (in pmol/mg, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>) compared to the theoretical value of 10363 pmol/mg. For example, the NiE fraction has a specific binding value of 3724 pmol/mg and hence 36% (3724/10363) of the total protein in NiE (0.92 mg) is functional NTS1 (0.331 mg).</p>4<p>The amount of functional NTS1 was estimated from data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>.</p>5<p>The diluted NiE is 91% functional after 6 hrs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t001" target="_blank">Table 1</a>). This decrease of the amount of functional NTS1 is indicated by a left pointing arrow. The corresponding amount of misfolded NTS1 is listed.</p>6<p>The amount of functional and misfolded NTS1 was calculated from the total NTS1 amount in the respective fractions considering the proportion of functional receptors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>). For example, the NiE contained 0.443 mg total NTS1 which is 80% functional<sup>4</sup> i.e. 0.355 mg functional NTS1 and 0.088 mg misfolded NTS1.</p>7<p>The amount of contaminants was calculated by subtracting the total NTS1 amount from the total protein content of a given fraction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t004" target="_blank">Table 4</a>).</p>8<p>The total protein content of the NiE, NTFT and NTE fractions was determined by the Amido Black method.</p
Purification of NTS1.
<p>The progress of purification was monitored by SDS-PAGE (NuPAGE 4–12% Bis-Tris gel, Invitrogen, 1× MES SDS buffer) and SimplyBlue staining. Lane M: Novagen Perfect Protein Marker (15–150 kDa); lanes SN, NiE and NTFT: 5 µg of protein per lane; lane NTE: 3 µg of protein. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012579#pone-0012579-t003" target="_blank">Table 3</a>.</p
Structural Dynamics and Thermostabilization of Neurotensin Receptor 1
The neurotensin receptor NTSR1 binds
the peptide agonist neurotensin
(NTS) and signals preferentially via the G<sub>q</sub> protein. Recently,
Grisshammer and co-workers reported the crystal structure of a thermostable
mutant NTSR1-GW5 with NTS bound. Understanding how the mutations thermostabilize
the structure would allow efficient design of thermostable mutant
GPCRs for protein purification, and subsequent biophysical studies.
Using microsecond scale molecular dynamics simulations (4 μs)
of the thermostable mutant NTSR1-GW5 and wild type NTSR1, we have
elucidated the structural and energetic factors that affect the thermostability
and dynamics of NTSR1. The thermostable mutant NTSR1-GW5 is found
to be less flexible and less dynamic than the wild type NTSR1. The
point mutations confer thermostability by improving the interhelical
hydrogen bonds, hydrophobic packing, and receptor interactions with
the lipid bilayer, especially in the intracellular regions. During
MD, NTSR1-GW5 becomes more hydrated compared to wild type NTSR1, with
tight hydrogen bonded water clusters within the transmembrane core
of the receptor, thus providing evidence that water plays an important
role in improving helical packing in the thermostable mutant. Our
studies provide valuable insights into the stability and functioning
of NTSR1 that will be useful in future design of thermostable mutants
of other peptide GPCRs
Dynamic Behavior of the Active and Inactive States of the Adenosine A<sub>2A</sub> Receptor
The adenosine A<sub>2A</sub> receptor
(A<sub>2A</sub>R) belongs
to the superfamily of membrane proteins called the G-protein-coupled
receptors (GPCRs) that form one of the largest superfamilies of drug
targets. Deriving thermostable mutants has been one of the strategies
used for crystallization of A<sub>2A</sub>R in both the agonist and
antagonist bound conformational states. The crystal structures do
not reveal differences in the activation mechanism of the mutant receptors
compared to the wild type receptor, that have been observed experimentally.
These differences stem from the dynamic behavior of the mutant receptors.
Furthermore, it is not understood how the mutations confer thermostability.
Since these details are difficult to obtain from experiments, we have
used atomic level simulations to elucidate the dynamic behavior of
the agonist and antagonist bound mutants as well the wild type A<sub>2A</sub>R. We found that significant enthalpic contribution leads
to stabilization of both the inactive state (StaR2) and active-like
state (GL31) thermostable mutants of A<sub>2A</sub>R. Stabilization
resulting from mutations of bulky residues to alanine is due to the
formation of interhelical hydrogen bonds and van der Waals packing
that improves the transmembrane domain packing. The thermostable mutant
GL31 shows less movement of the transmembrane helix TM6 with respect
to TM3 than the wild type receptor. While restricted dynamics of GL31
is advantageous in its purification and crystallization, it could
also be the reason why these mutants are not efficient in activating
the G proteins. We observed that the calculated stress on each residue
is higher in the wild type receptor compared to the thermostable mutants,
and this stress is required for activation to occur. Thus, reduced
dynamic behavior of the thermostable mutants leading to lowered activation
of these receptors originates from reduced stress on each residue.
Finally, accurate calculation of the change in free energy for single
mutations shows good correlation with the change in the measured thermostability.
These results provide insights into the effect of mutations that can
be incorporated in deriving thermostable mutants for other GPCRs
Optimization of NTS1 expression under different induction conditions using a stable T-REx-293 cell line.
<p>The data are collected from a selected high-expressing clone. Cells were grown in suspension in CD OptiCHO medium supplemented with 4 mM L-glutamine and 1% certified FBS and were induced in the late exponential growth phase (at a viable cell density of 2 million cells/ml) with tetracycline. The addition of sodium butyrate enhanced expression levels. Intact cells were subjected to [<sup>3</sup>H]NT binding assay to determine the number of receptors located at the cell-surface. For all conditions, n = 2, error bars indicate SEM (standard error of the mean).</p
Thermostabilization of the β<sub>1</sub>‑Adrenergic Receptor Correlates with Increased Entropy of the Inactive State
The
dynamic nature of GPCRs is a major hurdle in their purification
and crystallization. Thermostabilization can facilitate GPCR structure
determination, as has been shown by the structure of the thermostabilized
β<sub>1</sub>-adrenergic receptor (β<sub>1</sub>AR) mutant,
m23-β<sub>1</sub>AR, which has been thermostabilized in the
inactive state. However, it is unclear from the structure how the
six thermostabilizing mutations in m23-β<sub>1</sub>AR affect
receptor dynamics. We have used molecular dynamics simulations in
explicit solvent to compare the conformational ensembles for both
wild type β<sub>1</sub>AR (wt-β<sub>1</sub>AR) and m23-β<sub>1</sub>AR. Thermostabilization results in an increase in the number
of accessible microscopic conformational states within the inactive
state ensemble, effectively increasing the side chain entropy of the
inactive state at room temperature, while suppressing large-scale
main chain conformational changes that lead to activation. We identified
several diverse mechanisms of thermostabilization upon mutation. These
include decrease of long-range correlated movement between residues
in the G-protein coupling site to the extracellular region (Y227A<sup>5.58</sup>, F338M<sup>7.48</sup>), formation of new hydrogen bonds
(R68S), and reduction of local stress (Y227<sup>5.58</sup>, F327<sup>7.37</sup>, and F338<sup>7.48</sup>). This study provides insights
into microscopic mechanisms underlying thermostability that leads
to an understanding of the effect of these mutations on the structure
of the receptor