50
2 Synthesis of In-Tether Chiral Center Peptides …
REMD simulations were carried out for 200 ns, achieving a total simulation time of
4.8 μs for each peptide.
2.4.7.2 Clustering Analysis, Calculated Helicity and Ramachandran
Plots
Clustering analysis was performed on 10,000 snapshots sampled at 300 K, using
the ‘gromos’ [70] method with 1Å cutoff based on RMSD of backbone and Cβ
atoms. The representative structures and population of major clusters of 7 peptides
were shown in Fig. 2.11. Calculated helicity (helical content) was defined as the
percentage of the structures with all residues in the right-handed-helix conformation
(−160°<φ<−40°, −80°<ψ<40°). The calculated helicity from REMD simulations
of the five peptides is shown in Table 2.7.
For each peptide, the Ramachandran plot was drawn using the φ, ψ values of all
five residues together, from 10,000 snapshots saved during the simulation. To draw
the plot, the whole 360° × 360° φ, ψ space is divided into 300 × 300 small bins, and
the relative probability of each bin was calculated from the counts. A logarithmic
color scale is used to visualize the probability distribution, and the bins not sampled
are left in white.
2.4.8 Molecular Cloning, Protein Expression,
and Purification
(a) Cloning, Expression, and Purification of ER- LBD4. Human ER- LBD 301553 was cloned into pET23b via NdeI and XhoI generating untagged constructs.
Expression was carried out in E. coli BL21 (DE3) without IPTG induce. Cultures
were grown in 2YT medium at 37°C to OD 600 of 0.8 and then transferred to 20°C
for 18 h. Cells were harvested by centrifugation and flash frozen. Harvested cells
were lysed via sonication in 100 mM lysis buffer (Tris-Cl pH 8.1, 300 mM KCl,
5 mM EDTA, 4 mM DTT, and 1 mmol/L PMSF). Cell debris were removed by
centrifugation and the supernatant ran over a 1 mL estradiol affinity column (PDI
technology) and the column was then eluted with elution buffer (100 μM estradiol,
20 mM Tris pH 8.1, and 0.25 M NaSCN). High molecular weight species and excess
salts were removed on a Superdex 200 column equilibrated in buffer of 50 mM Tris
pH 7.4, 150 mM NaCl, 10% glycerol, and 1 mM DTT.
Primer sequence:
ER-alpha-NdeI-301: GTGTACACATATGtctaagaagaacagcctggccttgt
ER-alpha-XhoI-553: Ccctcgagttaagtgggcgcatgtaggcggt
2 Synthesis of In-Tether Chiral Center Peptides …
REMD simulations were carried out for 200 ns, achieving a total simulation time of
4.8 μs for each peptide.
2.4.7.2 Clustering Analysis, Calculated Helicity and Ramachandran
Plots
Clustering analysis was performed on 10,000 snapshots sampled at 300 K, using
the ‘gromos’ [70] method with 1Å cutoff based on RMSD of backbone and Cβ
atoms. The representative structures and population of major clusters of 7 peptides
were shown in Fig. 2.11. Calculated helicity (helical content) was defined as the
percentage of the structures with all residues in the right-handed-helix conformation
(−160°<φ<−40°, −80°<ψ<40°). The calculated helicity from REMD simulations
of the five peptides is shown in Table 2.7.
For each peptide, the Ramachandran plot was drawn using the φ, ψ values of all
five residues together, from 10,000 snapshots saved during the simulation. To draw
the plot, the whole 360° × 360° φ, ψ space is divided into 300 × 300 small bins, and
the relative probability of each bin was calculated from the counts. A logarithmic
color scale is used to visualize the probability distribution, and the bins not sampled
are left in white.
2.4.8 Molecular Cloning, Protein Expression,
and Purification
(a) Cloning, Expression, and Purification of ER- LBD4. Human ER- LBD 301553 was cloned into pET23b via NdeI and XhoI generating untagged constructs.
Expression was carried out in E. coli BL21 (DE3) without IPTG induce. Cultures
were grown in 2YT medium at 37°C to OD 600 of 0.8 and then transferred to 20°C
for 18 h. Cells were harvested by centrifugation and flash frozen. Harvested cells
were lysed via sonication in 100 mM lysis buffer (Tris-Cl pH 8.1, 300 mM KCl,
5 mM EDTA, 4 mM DTT, and 1 mmol/L PMSF). Cell debris were removed by
centrifugation and the supernatant ran over a 1 mL estradiol affinity column (PDI
technology) and the column was then eluted with elution buffer (100 μM estradiol,
20 mM Tris pH 8.1, and 0.25 M NaSCN). High molecular weight species and excess
salts were removed on a Superdex 200 column equilibrated in buffer of 50 mM Tris
pH 7.4, 150 mM NaCl, 10% glycerol, and 1 mM DTT.
Primer sequence:
ER-alpha-NdeI-301: GTGTACACATATGtctaagaagaacagcctggccttgt
ER-alpha-XhoI-553: Ccctcgagttaagtgggcgcatgtaggcggt