Computational Methods to Study Protein Structure and Dynamics - Peter Hildebrand Universität Leipzig
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Computational Methods to Study Protein Structure and
Dynamics
Peter Hildebrand
Universität
Leipzig
Computational Methods to Study Protein Structure and
Dynamics
Date Program
10-08-2020 The world is dynamic: protein flexibility, specifics of membrane proteins,
GPCRs like to move it: activation cycle, microswitches, coupling specificity,
techniques to monitor GPCR molecular dynamics
Practical part: Visualization of MD trajectories #1: MDsrv,
Setup MD simulations #1: Preparation of protein model (model missing parts,
remove stabilizing mutations, modifications), setup with CHARMM-GUI #1
12-08-2020 MD simulations: Theory and techniques: physical principles in a nutshell,
simulation packages, force fields, parametrization
Practical part: Setup MD simulations using CHARMM-GUI #2, Analysis of MD
trajectories #1 (GROMACS, VMD)
14-08-2020 MD simulations: application and limitations, sampling techniques, prominent
examples
Practical part: Analysis of MD trajectories #2, Visualization of MD trajectories #2
17-08-2020 Introduction into the Rosetta MC simulation program. De novo structure
prediction and the CASP experiment. The loop closure problem and
comparative modeling with Rosetta.
Practical part: de novo structure prediction and comparative modeling with
Rosetta
19-08-2020 Integrative structural biology with Rosetta. Protein structure prediction from
limited data: X-ray crystallography, Cryo-Electron Microscopy, NMR-
spectroscopy, EPR-spectroscopy, mass spectrometry
Practical part: Integrative structural biology with Rosetta
21-08-2020 Protein design with Rosetta. Design of antibodies, immunogens, enzymes,
protein and ligand binders, multi-state design
Practical part: Protein design with Rosetta
Key questions in GPCR research
out
LIGAND
ACTIVATION GPCR
More than 1/3 of
all drugs bind to
GPCRs! SPECIFICITY
GDP/GTP
EXCHANGE
G-protein
in
Proteins like to move it
Proteins….
DAY 1
….like to move it
Proteins have been perceived as rigid
From Cherezov et al. Nature, 2007 From Scheerer et al. Nature, 2008
….because X-ray yields static pictures
Dance of the proteins ΔG = ΔH - T*ΔS
The dynamic world of membrane proteins
BASICS OF PROTEIN STRUCTURE
Several of the following slights were kindly provided by Dr. Martin Heck
from Charité Berlin
Proteins like to move it
proteins are biopolymers
Proteins
NH
R
O
NH
R Amino acids
O
NH
R
O
Peptide bond
NH
(=Amid)
R
O
Proteins like to move it
1-, 3-letter codes of amino acids
3-letter 1-letter 3-letter 1-letter
amino acids -code -code amino acid -code -code
Alanine Ala A Leucine Leu L
Lysine Lys K
Arginine Arg R
Methionine Met M
Asparagine Asn N
Phenylalanine Phe F
Aspartate Asp D
Proline Pro P
Cysteine Cys C
Serine Ser S
Glutamine Gln Q
Threonine Thr T
Glutamate Glu E
Tryptophan Trp W
Glycine Gly G
Tyrosine Tyr Y
Histidine His H
Isoleucine Ile I Valine Val V
Proteins like to move it
Titeltext
proteins are biopolymers
Gly
Trp
Leu
Ala
Thr
Val
Ala
Ser
Lys
Glu
Glu
Pro
Thr
Main and Main chain schematic
Sequence side chains ‚backbone‘ ‚Cartoon‘
(segment) ‚sticks‘
Proteins like to move itTiteltext Proteines: hierarchy
Primary Secondary Tertiary Quaternary
structure structure structure structure
Glu
Glu
Lys
Ser
Ala
covalent bonds Sements are Chain folds to Complex of
(sequence) folded a 3D several subunits
structure
Proteins like to move itTiteltext Secondary structures
Secondary structure:
Folded parts of the polypeptide chain
α-Helix β-sheet
Stabilized through side chains of main chain
Proteins like to move itTiteltext Secondary structure: α-helix
•The C=O of a residue (i) is
hydrogen bonds to the N-H
group of the residue (i+4)
•3.6 residues per winding
•1.4 Å (0,14 nm) shift / residue
•5.4 Å pitch / winding
Proteins like to move itTiteltext Secondary structure
Amino acid properties
determine structure
α-Helix β-sheet
Glycine
Tyrosine Relative propensity of an
Serine amino acid
Valine
Leucine
Alanin
Glutamate
A single point mutation can significantly change a proteins structure /
function
Proteins like to move itTiteltext Secondary structure: α-Helix
Protein with high α-content:
soluble proteins Membrane proteins
Heptahelical
receptors
Hämoglobin GPCR
Proteins like to move itTiteltextG-Protein coupled Rezeptors (GPCR)
3-4 % of genes of the human genom encode > 1000 GPCRs
light/retinal purine Adrenaline Dopamine
rhodopsin A2A adrenoceptor D3
Proteins like to move itTiteltextThe RCSB Protein Data Bank (PDB)
http://www.rcsb.org/
Proteins like to move itClassification of amino acids by chemistry
-
COO
+
H3N C H
R
● Property of side chain:
- non polar (hydrophobic)
- aromatic
- polar, uncharged
- basic
- acidic
Proteins like to move itClassification of amino acids by chemistry
Amino acids with non polar side chains
- - - -
COO COO COO COO
+ + + +
H3N C H H3N C H H3N C H H3N C H
CH3 H3C CH CH2 H3C CH
CH3 H3C CH CH2
CH3 CH3
Alanine Valine Leucine Isoleucine
Proteins like to move itClassification of amino acids by chemistry
Titeltext
Amino acids with aromatic side chains
- - -
COO COO COO
+ + +
H3N C H H3N C H H3N C H
CH2 CH2 CH2
N
H
OH
Phenylalanine Tyrosine Tryptophan
Proteins like to move itTiteltextFunction of hydrophobic amino acids
● Stabilization of protein structures by
hydrophobic interactions
● Anchoring of proteins in
membranes
● building hydrophobic binding pockets for
hydrophobic substrates
● Hydrophobic collapse during folding
Proteins like to move itClassification of amino acids by chemistry
Titeltext
Amino acids with polar, uncharged side chains
- - - -
COO COO COO COO
+ + + +
H3N C H H3N C H H3N C H H3N C H
CH2 HC OH CH2 CH2
OH CH3 C O CH2
NH2 C O
NH2
Serine Threonine Asparagine Glutamine
Proteins like to move itClassification of amino acids by chemistry
Titeltext
Amino acids with acidic side chains
- -
COO COO
+ +
H3N C H H3N C H
CH2 CH2
-
COO CH2
COO -
Aspartate Glutamate
(Aspartic acid) (Glutamic acid)
Proteins like to move itClassification of amino acids by chemistry
Amino acid with alkaline side chain
- - -
COO COO COO
+ + +
H3N C H H3N C H H3N C H
CH2 CH2 CH2
CH2 CH2
+
CH2 CH2 HN NH
CH2 NH
+
NH3 C +
H2N NH2
Lysine Arginine Histidine
Proteins like to move itClassification of amino acids by chemistry
Amino acid with alkaline side chain
- -
COO COO
+ +
H3N C H H3N C H
CH2
His CH2
+
HN NH N NH
in equilibrium at physiological conditions
Proteins like to move itClassification of amino acids by chemistry
Special amino acids
- COO
-
COO
+ +
H3N C H H2N C H
H H2C CH2
CH2
Prolin
Glycin
-’destabilize secondary structures (proline
termintes alpha helices
-induce tertiary structure flexibility
Proteins like to move itTiteltext Amide
O O
C C
R NH2 R NH R
Amide:
● from carbon acids und amines
● can be hydrolyzed
● forms hydrogen bonds
● partially double bond of the C-N bond (mesomerie)
● neutral
Proteins like to move itExercise 1 (also possible: two people)
Titeltext
Three peptide:
● draw a peptide from three different residues
● show rotatable bonds
● show atoms capable of forming hydrogen bonds
● discuss polarities of residue
Proteins like to move itMesomeric structure of amide (Peptide bond)
Titeltext
_
O O
+
C N C N
H H
partially double bond of the C-N bond (mesomeric)
● neutral
● not rotatable
Proteins like to move itTiteltext Structure of Polypeptide
● ω = the C-N bond, not rotatable
● ϕ = rotatable N-C bond
● ψ = rotatable C-C bond
https://commons.wikimedia.org/wiki/File:Ramaplot.png
Proteins like to move itExtended structure of polypeptide chain
Titeltext
Peptides are rigid and not flexible here
O R O R O R O R
H H H H
Polypeptide N N N N N N N N
H H H H
R O R O R O R O
Side chain
Main chain
Side chain
Proteins like to move itTiteltextConformational changes of proteins
Conformational change: rotation of C-C single bonds result in
different relative arrangement of atoms
Conformational changes in:
● folding and denaturation
● miss-folding (e.g. prions)
● movement (e.g. muscle)
● catalysis (enzyme)
Proteins like to move itTiteltextConformational changes of proteins
Denaturation
heat
pH-value
salt
metal ions
Proteins like to move itTiteltextConformational changes of proteins
On hydrogen bonds On salt bridges
H
H N
N + -
CH2 NH OOC CH2
N H O CH2
Ser
CH2
CH2 Glu
His
His
protonation Deprotonation
H
H H N
N
+ O N COO
-
NH CH2
CH2
Ser CH2 CH2
CH2
His His
Glu
Proteins like to move itThe dynamic world of membrane proteins
SPECIFICS OF MEMBRANE PROTEIN STRUCTURE
Proteins like to move itTiteltext Hydrophobicity
• membrane bilayer thickness:
0,3-0,4 nanometer
• hydrophobic ‚carbon‘ core
• hydrophilic interface: lipid
headgroups and water
• unisotropic environment
Bowie, Nature 2005,
Solving the membrane protein folding problem
Proteins like to move itFree energies transfer of amino acids
Titeltext
from water to octanol
• charged residues: green
• polar residues: yellow
• hydrophobic residues: purple
Bowie, Nature 2005,
Solving the membrane protein folding problem
Proteins like to move itExercise 2: Secondary structure prediction
Titeltext
Find the seven transmembrane helices of rhodopsin
Proteins like to move itTiteltext Hydrophobicity plot
Hydrophobicity can be used to predict transmembrane segments
Proteins like to move itHigh variety of different hydrophobicity scales
Titeltext
Simm et al. 2016:50 years of amino
acid hydrophobicity scales:
revisiting the capacity for peptide
classification
Proteins like to move itTiteltext
Killian et al.,TIBS 2000 Rose et al., NAR, 2009,
Proteins like to move itTiteltext Hydrophobicity plots
https://web.expasy.org/protscale/
Proteins like to move itSecondary structure / topology prediction
Titeltext
- DAS-TMfilter based on:
- HMMTOP - solvent accessibility
- MARCOIL - secondary structure
- PHOBIUS - signal peptides
- PREDICTPROTEIN - positive inside rule
- SOSUI - hydrophobicity
- TMHMM Algorithms:
- TMpred - Markov Modeling
- TopPred - Neuronal Networks
- UniProt/Swiss-Prot - Machine Learning
- Rhythm
Proteins like to move itTiteltext Two stages of membrane protein folding
• Nascent polypeptide chain
inserted into bilayer by
translocon
• Transmembrane helices
laterally exit translocon
channel
• Transmembrane helices
Bowie, Nature 2005, assembly forming a
Solving the membrane protein
folding problem membrane protein
Proteins like to move itA three stage model of folding
Subunit A-C:
Encoded by the
mitochondrial
1 3
2 DNA
Subunit D-M:
Encoded by the
nucleus DNA
3
3
Idea from
Popot, 1990
Proteins like to move itBacteriorhodopsin (1c3w) seen from a lipid angle
O and N, without
h-bonding partner
in the crystal
Aromats
27 Å
C, or h-bonded
O and N Lipids
Proteins like to move itGly-helixcaps
• Helices are capped
at their termini
• Stabilize helix end
• Alternative h-bonds
C3 C2 ‚C‘‘ and C‘‘‘ are main chain
C‘‘‘ hydrogen bonded to C3 and
thus polar in αL motifs‘
C‘‘
C1
Aurora & Rose, Protein Sci. 1998
C‘= GlyHydrophobic Gly caps in transmembrane helices
Aromatic 33% g (polar C‘‘ and/or C‘‘‘) Δ N-term
belt 66% G (apolar C‘‘ and C‘‘‘)
Hildebrand, Preissner & Frömmel, FEBS Letters 2004Hydrophobicity: hydrophobic mismatch
Titeltext
• hydrophobic region (blue) of
protein is thicker than the
bilayer hydrocarbon core:
• - the protein can thin
- or the bilayer can thicken
Bowie, Nature 2005,
Solving the membrane protein folding problem
Proteins like to move itHelix-helix packing: different packing motifs
• Left-handed packing:
Left right
inter-digitation of side-chains
small packing angles
• right-handed packing:
close packing of main-chains
larger packing angles
Cβ
Cα
Cα
Hildebrand et al., Protein, 2005,
Analysis and prediction of helix-helix interactions
in membrane channels and transporters
Proteins like to move itHelix-helix packing: different packing motifs
Titeltext
Right-handed A
• Left-handed packing:
left-handed
C gate C
D
E E
heptad repeat of large
coil F
G G residues
H
I
I
K
LxxxLxxL (Large)
L L
M
N N
P • right-handed packing:
Q Q
R
S
octad repeat of small
S
T
V V residues
W
Y Y SxxxSxxxS (Small)
-2 -1 0 1 2
propensities for buried amino acids Hildebrand et al., Protein, 2005,
Analysis and prediction of helix-helix interactions
in membrane channels and transporters
Proteins like to move itMolecular packing and packing defects
Packing density:
VVdW/VVdW+Vsol
14*Membrane
13*Channels -Coils
Vsolv Solvent accessible
VVdWVan der Waals
Hildebrand et al, Biophys J. 2005
Proteins like to move itMolecular packing and packing defects
Packing density:
VVdW/VVdW+Vsol
14*Membrane
13*Channels -Coils
Hildebrand et al, Biophys J. 2005
Proteins like to move itMolecular packing and packing defects
hydrophobic
hydrophilic
cavities
glycerol-3-phosphate transporter (Protein Data Bank code 1pw4)
Hildebrand et al, Biophys J. 2005
Proteins like to move itTiteltext Curvature: elastic energy
• two types of lipid:
cylindrical or cone-shaped
• a, cone-shaped lipid: causes
leaflets to curve away from
one another.
• b, forcing them into a bilayer
causes overpacking in the
hydrocarbon tails.
• c, hour-glass-shaped
protein releases some of this
stored curvature elastic
energy.
Bowie, Nature 2005,
Solving the membrane protein folding problem
Proteins like to move itStabilized by
Stabilizing elements Loops
Interaction of
helixcaps in the
polar milieu
Interaction with
the polar lipid
headgroups
‚Lateral pressure‘
of the membran
Interactions with
ligands
interactions of
helices in the
lipophilic milieuThe dynamic world of membrane proteins
G PROTEIN COUPLED RECEPTORS
Proteins like to move itHepta helical receptors
Titeltext G-Protein coupled Rezeptors (GPCR)
3-4 % of genes of the human genom encode > 1000 GPCRs
Licht/Retinal Purin Adrenalin Dopamin
Rhodopsin A2A Adrenoceptor D3
Proteins like to move itG Protein Coupled Receptors
Biogenic amines (adrenaline), peptides (angiotensin), lipids (cannaboids),….
GPCR
~ 800 different human GPCRs couple to
one or several G proteins
(Gs, Gi, Gq,G11/12) / arrestins (1-4)
- involved in cancer, cardiac diseases,
Arr
Gi Gs obesity, Alzheimer,…
Cellular response e.g.: Recent developments in biased agonism
Wisner et al. & Lefkowitz, Curr Opin Cell Biol, 2014
Proteins like to move itTiteltextGPCR respond to (extracellular) signals
A Biogenic amines (adrenalin), peptides (angiotensin), lipids (cannaboids),….
R R*
E γ α γ α E1 γ β E2
β GDP β α GTP
GTP GDP
second
B messenger
GTP GDP
hν
γβ GDP
α γ β α γ β GTP
α
PDE PDE
From Klaus-Peter Hofmann
Proteins like to move itGPCRs
Chemistry 2012
OUT
RECEPTOR
Bob Lefkowitz and Brian Kobilka
G-protein
IN
Proteins like to move itTM6 moves outwards during receptor activation
GαCT ( = Gα C-terminal helix) replaces Holo-Gαβγ
R / R*
inactiv
activ
Scheerer, Park, Hildebrand, Kim, Krauss, Choe, Park, Hofmann & Ernst, Nature 2008
Hofmann, Scheerer, Hildebrand, Choe, Park, Heck & Ernst, TIBS 2009
Proteins like to move itActivation (and regeneration cycle) of rhodopsin
Titeltext
Photoactivation Activation
Batho
ns
Lumi
Light µs
ener ms
gy Meta I Meta II forms
min all-trans-retinal
Opsin apoprotein / Ops*
min 11-cis-retinal
Rhodopsin
Hofmann, Scheerer, Hildebrand, Choe, Park, Heck & Ernst, TIBS 2009
Proteins like to move itActivation (and regeneration cycle) of rhodopsin
Titeltext
in
out
Rhodopsin Bathorhodopsin Lumirhodopsin Metarhodopsin II Opsin
1f88.pdb 3g87.pdb 3g87.pdb 3pxo.pdb 3cap.pdb
1u19.pdb
1gzm.pdb
Proteins like to move itmovie M2 activation
Static snapshots
Snapshopts fromstructure
from crystal crystal analysis
structures
- Micro-switches promote fast and concerted motions during activation
Arg 1353.50 Tyr 2235.58 Tyr 3067.53
(E(D)RY motif) (Y(x)7KR motif) (NPxxY motif)
Rhodopsin MII / opsin MII●GtαCT
inactive active active and
Scheerer, Park, Hildebrand, Kim, Krause, Choe, Park, Hofmann & Ernst, Nature 2008
coupled
Leipzig-Dresden Bioscience Meeting 27.02.2019Titeltext Aktivierung eines Rezeptors
inaktiv
aktiv
aus Scheerer et al.
Nature 2008 Ramon Guixa
Proteins like to move itReceptor activation
GαCT ( = Gα C-terminal helix) mimics Holo-Gαβγ
TM6
Ramon Guixa
Proteins like to move it...the problem with (crystal) structures
Titeltext
779 receptors but
only 44 resolved
(Xiang et al. TIPS 2016)
GPCR Network: http://gpcr.scripps.edu
Proteins like to move itTiteltext Loopmodelling
Proteins like to move itSuperLooper, An Interactive webtool
Proteins like to move itSuperLooper, An Interactive webtool
What we have: What we want:
What we need:
Database & Search algorithm
huge efficient
Suitable as web application
Proteins like to move itTiteltext SuperLooper, An Interactive webtool
SL2 – Database
• 114,693 structures
• 901,609,231 fragments
• 3-35 residue length
Proteins like to move itSuperLooper, An Interactive webtool
SL2 – Search algorithm / criteria
1. Extract fragments with sequence length of missing fragment
2. Weight fragments according to sequence similarity
3. Rank fragments according to geometrical fingerprint
matching (RMSD > 0.75 A)
4. Sort similar fragments with backbone RMSD < 0.5 A of
top-1000 list out
5. Find clashes
6. Present results to user
Proteins like to move itTiteltext SuperLooper, An Interactive webtool
Geometrical fingerprint
Proteins like to move itTiteltext SuperLooper, An Interactive webtool
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