9/13/17 It’s All About Science and Math
Undergraduate Open House
College of Sciences
2 Volunteers/day
12 Noon – 1 pm
Friday | Sep 15
Friday | Sep 29
Boggs 2nd Floor
The PKC-Flavones Study:
Molecular Modeling
CHEM 4582
Fall 2017 | Dr. Mary E. Peek
Teaching and Learning Goals
§
§
§
Develop a structural basis for a flavonoid’s impact on PKC
activity
Develop expertise with PyMOL
Gain experience using AutoDock VINA
1 9/13/17 What is molecular modeling?
A theoretical or computer-based approach for
describing on a molecular level how biological
macromolecules behave
Build
Models
Energy
Minimization
Molecular
Dynamics
Quantum
Mechanics
Docking is a type of energy
The Potential
Energy
Function
minimization
experiment
Ubond = oscillations about
the equilibrium bond length
University of Illinois, Urbana-Champaign
Uangle = oscillations of 3 atoms about an equilibrium bond angle
Udihedral = torsional rotation of 4 atoms about a central bond
Unonbond = non-bonded energy terms (electrostatics and Lenard-Jones)
2 9/13/17 ENERGY
University of California, Berkeley
GOAL
Find the
global
minimum
GEOMETRY
Experimental Overview
Edit Protein
File
Download
PDB File
+
Generate
Flavonoid
Structure
Energy
Minimize
Flavonoid
Run
AutoDock
Vina
1. Find the active site of PKC
2. Analyze how the flavonoid can bind to PKC
3. How tightly does the flavonoid bind to PKC
based on the computed Kd value?
3 9/13/17 Based on your analysis of the PKCflavonoid complex,
propose a structural basis for the
impact of your particular flavonoids
on PKC activity
Get PDB Files from the Protein Data
Bank
www.rcsb.org
4 9/13/17 Looking at a Protein Data Bank File
• Header Information and Remarks
• SEQRES
amino acid or nucleotide sequence
• HETNAM
name of any heteroatoms present
i.e. any non-C, N, O, P, S
Chain
Residue
#
AA
ATOM
ATOM
ATOM
ATOM
1
2
3
4
N
CA
C
O
ILE A
ILE A
ILE A
ILE A
16
16
16
16
-8.155
-8.150
-9.405
-10.533
Occupancy
9.648
8.766
9.018
8.888
20.365
19.179
18.348
18.870
Thermal
Factor
1.00 10.68
1.00 10.68
1.00 10.68
1.00 10.68
X, Y, Z Coordinates
Brief Introduction to PyMOL
5 9/13/17 PKC Reaction
O
O P O
Peptide or protein
substrate
Used
Phosphatidylserine
instead
PKC
ATP
DAG
Mg2+,Ca2+
Phosphorylated peptide
or protein substrate
ADP
DAG
Mg2+,Ca2+
Given this reaction,
where would the peptide bind to PKC?
Ligand File Preparation
6 9/13/17 Protein File Preparation
Structure of an EGCG-PKC Complex
Notice where
binding occurs
OH
OH
HO
O
OH
O
OH
OH
O
OH
OH
Epigallocatechin Gallate
(EGCG)
Any other
observations?
7 vina --receptor proteinname.pdbqt --ligand ligandname.pdbqt --center_x 43.679 --center_y
72.68 --center_z 43.374 --size_x 64 --size_y 100 --size_z 68 --out newoutputname.pdbqt
f.
Press Return. You will eventually see the screen shot in Figure 2.
9/13/17 g. Analyze the data.
If docking can occur at multiple
locations, then show representatives
N-terminal
Domain
Substrate
Binding Site
C-terminal
Domain
Figure 1. Screenshot of the output from moving to the Vina folder.
Output Screen Shot
Figure 2. Screen shot illustrating the initial response when a docking search is conducted, the
Affinity
ΔG°!
Convert
to example,
an actualonly
Kd value
percentage completion
overhere
time,means
and the
results.
In this
three binding modes were
obtained from the search space defined.
8 9/13/17 Binding Affinity in Biochemistry
PROTEIN PROTEIN
+ LIGAND
+ LIGAND
[PROTEIN-LIGAND]
[PROTEIN-LIGAND]
[PROTEIN] [LIGAND]
Keq =
Keq =
PROTEIN-LIGAND
PROTEIN-LIGAND
[PROTEIN] [LIGAND]
Keq = eΔG/(-RT)
ΔG = -RT ln (Keq)
R = 0.00195 kcal x mol-1 x K-1
T = 25°C = 298 K
ΔG = -RT ln (Keq)
Binding Affinity = Kd
-1
1-1
R = 0.00195 kcal x mol
Kd = x ΔK
e G/(-RT)
Keq = eΔG/(-RT)
T = 25°C = 298 K
Binding Affinity in Biochemistry
Binding Affinity = Kd
1
Kd =
eΔG/(-RT)
9 9/13/17 Structural Basis for Inhibition or
Enhancement
• INHIBITION
• ENHANCEMENT
– What is it about the look
of the complex that might
explain why the
flavonoid inhibits PKC
activity?
– What is it about the look
of the complex that might
explain why the
flavonoid enhances PKC
activity?
– Does the location of the
binding of the flavonoid
to PKC shed light on
why it inhibits activity?
– Does the location of the
binding of the flavonoid
to PKC shed light on
why it enhances activity?
Assessment #2 Due Electronically
• Watch Group Project Survival Guide
T-Square>Resources>Group Projects
• Complete the Conflict Mgt Style Quiz
T-Square>Resources>Group Projects
• Complete Assessment #2 by Sep 18 at 11:55 pm
T-Square>Tests & Quizzes>Assessment #2
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