Protein folding, stability and dynamics
❑ Revisions
❑ Protein folding
❑ Protein stability
❑ Protein dynamics
Outline
2Protein folding, stability and dynamics
Revisions: molecular interactions
3Molecular interactions
❑ Covalent interactions
▪ sharing of electrons
▪ under standard condition – very stable
▪ primary structure of proteins
❑ Non-covalent (weak) interactions
▪ electrostatic interactions
▪ polar interactions
▪ non-polar interactions
▪ secondary, tertiary and quaternary structure of proteins
Revisions: electrostatic interactions
4Molecular interactions - electrostatics
❑ Charge-charge interactions
▪ charged residues – Arg, Lys, Glu, Asp and His (low pH)
▪ long-range interaction – decrease with r2
▪ environment dependent
▪ permitivity (ε):
▪ 1 – vacuum
▪ 2-20 – interior of proteins, membranes
▪ 80 – bulk water -> water shields the chares form each
others
▪ salt concentration – counter ions close to charged residues
▪ pH – change in charge of molecule (His)
Revisions: polar interactions
5Molecular interactions - polar
❑ Hydrogen bonds (H-bonds)
▪ donor and acceptor atoms sharing hydrogen
▪ polar residues – Ser, Thr, Asn, Gln, Cys, Trp, Tyr and His (high pH)
▪ charged residues – Arg, Lys, Glu, Asp and His (low pH)
▪ governs formation of secondary structure
▪ H-bond distance: 2.8-3.4 Å
acceptor donor
Revisions: polar interactions
Molecular interactions - polar
❑ Aromatic (π-π) interactions
▪ attractive interaction between aromatic rings
▪ aromatic residues – Phe, Trp, Tyr and His
▪ distance of centre of mass – about 5 Å
sandwich
parallel
displaced
T-shaped
Revisions: non-polar interactions
7Molecular interactions – non-polar
❑ van der Waals (vdW) interactions
▪ between any two atoms -> all residues
▪ short-range interactions
▪ negligible beyond 5 Å
▪ tertiary structure
Revisions: non-polar interactions
8Molecular interactions – non-polar
❑ Hydrophobic interactions
▪ hydrophobic residues – Phe, Pro, Met, Leu, Ile, Val, Ala, and
possibly also Tyr and Trp
▪ entropic origin – water molecules ordered around hydrophobic
residues -> unfavorable
▪ hydrophobic packing -> release of some ordered water ->
favorable increase of entropy
▪ tertiary structure
❑ Levinthal’s paradox
❑ Anfinsen’s thermodynamic hypothesis
❑ Mechanisms of protein folding
❑ Energetics of protein folding
❑ Database of protein folding
Protein folding
9Protein folding
❑ Cyrus Levinthal
▪ 1968 – impossibility of random folding
▪ random folding
▪ 100 residue protein (average sized)
▪ 3 conformation per residue (many more)
▪ 0.1 ps sampling time per conformation (much longer)
▪ folding time = 3100*10-13 s ≈ 5*1034 s ≈
▪ 1 634 251 397 552 039 990 billions of years
❑ Experimental folding rates
▪ 1 ms to 10 min
Levinthal’s paradox
10Protein folding – Levinthal’s paradox
❑ Christian Anfinsen
▪ 1973 – protein folding in vitro
▪ refolding of ribonuclease
❑ Findings
▪ native structure of a protein is the thermodynamically stable
structure
▪ folding depends only on the amino acid sequence and on the
conditions of solution, and not on the kinetic folding route
Anfinsen’s thermodynamic hypothesis
11Protein folding – Anfinsen’s thermodynamic hypothesis
Thermodynamic and kinetic stability
Thermodynamic stability Kinetic stability
𝑁𝑎𝑡𝑖𝑣𝑒
𝐾 𝑒𝑞
𝐷𝑒𝑛𝑎𝑡𝑢𝑟𝑒𝑑 𝑁𝑎𝑡𝑖𝑣𝑒 →
𝑘
𝐷𝑒𝑛𝑎𝑡𝑢𝑟𝑒𝑑
Protein folding – Thermodynamic and kinetic stability 12
Mechanisms of protein folding
13Protein folding – mechanisms
❑ Nucleation-growth (propagation) model
▪ continuous growth of tertiary structure from initial nucleus of local
secondary structure
▪ it did not account for folding intermediates -> model dismissed
Mechanisms of protein folding
14Protein folding – mechanisms
❑ Framework model
▪ secondary structure folds first -> coalescence of secondary
structural units to the native protein
❑ Hydrophobic collapse model
▪ compaction of the protein -> folding in a confined volume ->
narrowing the conformational search to the native state
❑ Nucleation-condensation model
▪ concerted & cooperative secondary and tertiary structure formation
▪ transition state resembles distorted form of the native structure
▪ the least distorted part called folding nucleus or molten globule
Mechanisms of protein folding
15Protein folding – mechanisms
❑ Free energy of folding (ΔGfold = ΔH - T.ΔS)
▪ protein more structured -> ΔS↓ – unfavorable
▪ solvent less structured -> ΔS↑ – favorable
▪ hydrophobic interactions are driving “force”
▪ more non-covalent interactions -> ΔH↓ – favorable
Energetics of protein folding
16Protein folding – energetics
Energetics of protein folding
17Protein folding – energetics
Energetics of protein folding
18Protein folding – energetics
Energetics of protein folding
19Protein folding – energetics
❑ Basics of protein stability
❑ Database of protein stability
Protein stability
20Protein stability
Basics of protein stability
21Protein stability – basics
❑ Tertiary structure of protein
▪ sum of non-covalent weak interactions vs conformational entropy
▪ folded protein = thermodynamic compromise
▪ folded protein marginally more stable than unfolded (10-80 kJ/mol)
❑ Tertiary structure of protein
▪ sum of non-covalent weak interactions vs conformational entropy
▪ folded protein = thermodynamic compromise
▪ folded protein marginally more stable than unfolded (10-80 kJ/mol)
▪ Weak interactions are frequently disrupted
▪ denaturation - disrupted bonds replaced by bonds with solvent
▪ dynamics - disrupted bonds reformed between protein atoms
Basics of protein stability
22Protein stability – basics
Database of protein stability
23Protein stability – database
❑ ProTherm
▪ https://www.iitm.ac.in/bioinfo/ProTherm/index.html
▪ set of 746 unique proteins and 311 proteins with mutants
▪ numerical data of thermodynamic parameters for wild type and
mutant proteins
❑ Data
▪ Gibbs free energy change, enthalpy change, heat capacity change,
transition temperature
▪ secondary structure and accessibility for wild type residues
▪ experimental conditions, methods and activity information
Database of protein stability
24Protein stability – database
Database of protein stability
25Protein stability – database
Database of protein stability
26Protein stability – database
Database of protein stability
27Protein stability – database
❑ FireProtDB
▪ https://loschmidt.chemi.muni.cz/fireprotdb/
▪ numerical data of thermodynamic parameters for wild type and
mutant proteins
▪ More than 16,000 experimental stability data on ~ 300 proteins
▪ Manual curation
❑ Data
▪ Gibbs free energy change, enthalpy change, heat capacity change,
transition temperature
▪ experimental conditions and methods
Database of protein stability
28Protein stability – database
Database of protein stability
29Protein stability – database
❑ Basics of protein dynamics
❑ Characteristics of protein motions
❑ Dynamics and protein function
❑ Approaches to study dynamics
❑ Databases of dynamics
❑ Protein dynamics in biology
Protein dynamics
30Protein dynamics
❑ Origin of dynamics – disruption of weak interactions by
▪ thermal kinetic energy (kb.T)
▪ binding interactions (ligands or other proteins) – induced fit
❑ Protein atoms fluctuates around their average positions
▪ in tightly packed interior – movement restricted
▪ near surface – movement promoted by solvent movements
▪ -> proteins considered as “semi-liquids”
Introduction to protein dynamics
31Protein dynamics – introduction
❑ Divisions of protein motions
Characteristics of protein motions
32Protein dynamics – characteristics of protein motions
Type of motion Moving moiety Functionality
Local atoms; side-chains
bond vibration; ligand flexibility; temporal
diffusion pathways
Medium-scale secondary structures
active site conformational changes; motion of
hinge; peptide bond rotation;
Large-scale domains
hinge facilitated domain movements;
allosteric transition
Global subunits helix-loop transition; folding/unfolding
❑ Fluctuations
▪ less than 1 Å
▪ local motions
❑ Collective motions
▪ 1-10 Å
▪ medium and large-scale motions
❑ Triggered conformational changes
▪ more than 10 Å
▪ global motions
Amplitudes of protein motions
33Protein dynamics – characteristics of protein motions
Time scales of protein motions
34Protein dynamics – characteristics of protein motions
❑ Time scales governed by local environment
▪ interior – motions coupled due to packing restraints
▪ surface – no coupling of motions
❑ Example: aromatic ring flipping
▪ can occur on ps time scale, but often observed on ms time scale
▪ aromatic residues -> hydrophobic -> inside protein -> tightly packed
▪ -> low probability of synchronized movement of surrounding atoms
▪ -> prolonged time scale
Time scales of protein motions
35Protein dynamics – characteristics of protein motions
❑ NMR spectroscopy
❑ High resolution X-ray crystallography
❑ Computational
▪ Normal mode analysis (NMA)
▪ Molecular dynamics (MD)
Approaches to study dynamics
36Protein dynamics – approaches to study dynamics
❑ Ensemble of possible low energy conformations
❑ Directly shows possible amplitudes of motion
❑ Limited applicability to larger proteins
❑ Does not describe
▪ very fast motions & transition states
▪ time scales & energetics of motions
NMR spectroscopy
37Protein dynamics – approaches to study dynamics
❑ Average low energy structure - more conformations:
▪ in one structure only if both are separated by barrier
▪ in multiple structures
High resolution X-ray crystallography
38Protein dynamics – approaches to study dynamics
❑ Average low energy structure - more conformations:
▪ in one structure only if both are separated by barrier
▪ in multiple structures
❑ Crystalline state
▪ non-native contacts
▪ artificially lower amplitudes of motions
❑ Range of fluctuations – B-factors
❑ Does not describe
▪ very flexible regions
▪ collectiveness of motions
▪ time scales & energetics of motions
High resolution X-ray crystallography
39Protein dynamics – approaches to study dynamics
❑ Principle
▪ motion of system as harmonic vibration around a local minimum
▪ Coarse-grained model, residues connected with springs
❑ Small number of low-frequency normal modes
▪ shows directionality, collectiveness and sequence of global motions
❑ Does not describe
▪ local movements
▪ amplitudes & time scales
▪ energetics of motions
Normal mode analysis
40Protein dynamics – approaches to study dynamics
❑ Principle
▪ physical description of interactions within system (force field)
▪ Newton’s laws of motions
▪ forces acting on all atoms due to all atoms
▪ small time-step ~ 2 fs
Molecular dynamics
41Protein dynamics – approaches to study dynamics
❑ Principle
▪ physical description of interactions within system (force field)
▪ Newton’s laws of motions
▪ forces acting on all atoms due to all atoms
▪ small time-step ~ 2 fs
Molecular dynamics
42Protein dynamics – approaches to study dynamics
❑ Principle
▪ physical description of interactions within system (force field)
▪ Newton’s laws of motions
❑ Provides information on energetics, amplitudes and time
scales of local motions on atomic level
❑ Does not describe
▪ slower large scale motions (> ms)
Molecular dynamics
43Protein dynamics – approaches to study dynamics
Introduction to structural biology 44
Molecular dynamics
❑ Classical MD
Introduction to structural biology 45
Molecular dynamics
❑ Classical MD
❑ Enhanced sampling
▪ Adaptive sampling
▪ Metadynamics
▪ Accelerated MD
▪ Umbrella sampling
❑ Classical MD
❑ Enhanced sampling
▪ Adaptive sampling
▪ Metadynamics
▪ Accelerated MD
▪ Umbrella sampling
❑ Coarse-grained molecular dynamics
Introduction to structural biology 46
Molecular dynamics
Analysis of interactions
Molecular dynamics
47Protein dynamics – approaches to study dynamics
Ligand transport
Interaction with membraneLigand conversion
❑ Molecular Dynamics Extended Library (MoDEL)
❑ Dynameomics
❑ Molecular Movements Database (MolMovDB)
❑ ProMode-Elastic
Databases of dynamics
48Protein dynamics – databases
❑ http://mmb.pcb.ub.es/MoDEL/
❑ >1,700 MD simulations of proteins representatives of all
monomeric soluble structures in PDB
❑ 10 ns trajectories from MD simulations
❑ Data
▪ pre-computed analysis of geometry, secondary structure, flexibility
and inter-residue contacts
▪ trajectory video
▪ downloadable trajectories
MoDEL
49Protein dynamics – databases
MoDEL
50Protein dynamics – databases
❑ www.dynameomics.org
❑ MD simulations of over 800 proteins
❑ Longer trajectories (> 31 ns)
❑ Data
▪ pre-computed analysis of RMSD, SASA, Phi-Psi, and contacts
▪ trajectory video
▪ visualization of individual snapshots
▪ downloadable trajectories on request
Dynameomics
51Protein dynamics – databases
Dynameomics
52Protein dynamics – databases
❑ http://www.molmovdb.org/
❑ Collection of over 178 molecular motions
❑ Based on morphing – interpolation of motion between two
experimental crystal structures
❑ Data
▪ classification scheme for molecular motions
▪ movements animations
▪ hinge identification, structural analysis tools, references to
experimental crystal structures
▪ downloadable morphs as multi-model PDB
MolMovDB
53Protein dynamics – databases
MolMovDB
54Protein dynamics – databases
❑ https://pdbj.org/promode-elastic
❑ Normal mode analysis of PDB data – on PDB database
❑ Data
▪ fluctuation of atoms and dihedral angles
▪ correlation between fluctuating atoms
▪ distance map between residues
▪ animation of fluctuating molecules
▪ displacement vectors
ProMode-Elastic
55Protein dynamics – databases
ProMode-Elastic
56Protein dynamics – databases
ProMode-Elastic
57Protein dynamics – databases
❑ Adenylate kinase
❑ Motor proteins
Protein dynamics in biology
58Protein dynamics – role in biology
❑ Biological processes
▪ catalyzes interconversion of ATP + AMP ⇔ 2 ADP
❑ Large conformational change
▪ 90 degrees rotation of whole domain, up to 30 Å amplitude
▪ induced by binding of ATP
▪ shielding of bound substrate from solvent
Adenylate kinase
61Protein dynamics – role in biology
Adenylate kinase
62Protein dynamics – role in biology
Motor proteins
63Protein dynamics – role in biology
❑ Biological processes
▪ myosin movement along actin filament
▪ kinesin movement along microtubule
❑ Motor head
▪ ATPase domain – binding of ATP
▪ linker domain – changes conformation upon ATP binding
❑ One step
▪ moves a motor head for about 160 Å
▪ moves an attached cargo for about 80 Å
Motor proteins
64Protein dynamics – role in biology
References
❑ Gu, J. & Bourne, P. E. (2009). Structural Bioinformatics, 2nd Edition,
Wiley-Blackwell, Hoboken.
❑ Petsko, G. A. & Ringe, D. (2004). Protein Stucture and Function, New
Science Press, London.
❑ Schwede, T. & Peitsch, M. C. (2008). Computational Structural Biology:
Methods and Applications, World Scientific Publishing Company,
Singapore.
❑ Daggett, V. & Fersht, A. R. (2003). Is there a unifying mechanism for
protein folding? Trends in Biochemical Sciences 28: 18-25.
❑ Dill, K. A. et al. (2008). The protein folding problem. Annual Review of
Biophysics 37: 289-316.
References