Interatomic Forces and Protein Structure

1
Interatomic Forcesand Protein Structure

2
Forces that stabilize protein structure

• Interactions between atoms within the protein
  chain
• Interactions between the protein and the solvent

3
Bond types in proteins

• Covalent bonds
• Hydrogen bonds
• Metal ligands
• Ionic interactions
• Disulfide bonds
• Non-bond interactions

4
Favourable conformations in polypeptides

• Covalent interactions establish the structural
  framework of the protein molecule, the chemical
  expression of primary structure
• Backbone conformation constrained by steric
  restrictions on ? and ? torsions
• Sidechain conformations are also constrained
• Favourable sidechain conformations depend on the
  sidechain and also on its neighbours.
• S-S Bonds between cysteine residues can form
  within proteins

5
(No Transcript)
6
Electrostatic Interactions

• Charged side chains in protein can interact
  favorably with an opposing charge of another side
  chain according to Coulombs law
• Examples of favorable electrostatic interaction
  include that between positively charged lysine
  and negatively charged glutamic acid.
• Salts have the ability to shield electrostatic
  interactions.

7
Charge-charge interactions

• Coulomb interaction between two ions
• At close range, Coulomb interactions are as
  strong as covalent bonds
• Their energy decreases with 1/r and fall off to
  less than kT at about 56 nm separation between
  charges
• In practice, charge-charge interactions have been
  shown to be chemically significant at up to 15 Å
  in proteins
• Small charged metal ions can act as positive
  charge in an ion pair

Mg-ATP
Salt Bridge
8
Hydrogen bonds
O
H
N
C

• Noncovalent chemical bond in which an
  electronegative atom (a hydrogen-bond acceptor)
  shares a hydrogen atom with an electronegative
  atom with a bound hydrogen
• Energy 10-40 kJ/mol
• Approximately 1.7-3 Å in length
• Strength varies with angle of hydrogen-bond
  interaction
• Individually, not very strong, but the large
  numbers of hydrogen bonds in regular secondary
  structures stabilize the framework of the protein

9
H-bonding in lysozyme
Hydrogen Bonds
10
Hydrogen bonds in protein structure
11
van der Waals forces

• Nonspecific forces between like or unlike atoms
• Decrease with r6
• approximately 1 kJ/mol
• r0 is the sum of van der Waals radii for the two
  atoms. Van der Waals forces are attractive forces
  when rgt r0 and repulsive when rlt r0.

12
Approximate Strengths of Interactions between
atoms
13
The hydrophobic force

• Observation
• Hydrophobic residues are buried while hydrophilic
  residues are on the outside.
• The exterior surface area of proteins can be up
  to 60 polar atoms
• Proteins fold to maximize their effectiveness as
  hydrogen-bonding partners to water

• Explanation
• When hydrophobic residues are exposed to solvent,
  the extended hydrogen bonding structure of water
  is disrupted
• Breaking hydrogen bonds in water is energetically
  unfavourable
• Water molecules reorient around the hydrophobic
  molecule, so that the least number of hydrogen
  bonds are sacrificed to accommodate it
• Burying hydrophobic residues releases water and
  increases entropy.

14
Packing of Globular Proteins

• Secondary structures pack closely to one another
  and also intercalate with extended polypeptide
  chains
• Most polar residues face the outside of protein
  and interact with solvent but may be buried if
  H-bonding and charge is satisfied
• Most hydrophobic residues face the interior of
  the protein and interact with each other thereby
  minimizing contact with water
• van der Waals volume is about 72-77 of the
  total protein volume about 25 is not occupied
  by protein atoms. These cavities provide
  flexibility in protein conformation and dynamics
• Random coil or loops maybe of importance in
  protein function (interacting with other
  molecules, enzyme reactions)

15
The Thermodynamics of Folding

• Folding of a globular protein is a
  thermodynamically favored process, i.e. ?G must
  be negative.
• The folding process involves going from a
  multitude of random-coil conformations to a
  single folded structure.
• The folding process involves a decrease in
  randomness and thus a decrease in entropy -?S and
  an overall positive contribution to ?G. This
  decrease in entropy is termed conformational
  entropy.
• An overall negative ?G a result of features
  that yield a large negative ?H or some other
  increase in entropy on folding.

?G ?H - T?S
16
Protein Folding No Net Enthalpic Contribution

• Formation of secondary structure is an enthalpy
  driven process
• Energy derived from the formation of many van
  der waals and h-bonding interactions as well as
  the alignment of dipoles overcomes the loss of
  entropy associated with the formation of the
  peptide backbone conformation.
• Formation of tertiary structure is enthalpically
  unfavorable
• Energy loss in the burying of ion-pairs (1
  kcal/mol) and the breaking of shorter, stronger
  H2O bonds.
• Though some energy is gained from van der waals
  packing, very little is gained from the formation
  of internal h-bonds because as many h-bonds with
  water are broken as are formed in the process of
  folding a protein.
• Free energy associated with solvation of an ion
  is -60 kcal/mol
• An ion will NOT be buried in the hydrophobic
  interior of a protein.

17
Protein Folding Entropy Driven Process

• Upon protein folding
• hydrophobic residues move to the interior of the
  protein
• caged H2O molecules are released
• Enthalpy is gained unfavorable (?H )
• entropy is also gained (?S ) extremely
  favorable
• Increase in entropy of water compensates for the
  loss of conformational entropy of the protein and
  drives the protein folding process

18
Free energy of folding

• Difference in energy (free energy) between folded
  (native) and unfolded (denatured) state is small,
  5-15 kcal/mol
• Enthalpy and entropy differences balance each
  other, and DG is a small positive number.
• Small DG is necessary because too large a free
  energy change would mean a very stable protein,
  one that would never change
• However, structural flexibility is important to
  protein function, and proteins need to be degraded

19
Protein Folding

• What are the forces that guide this process?
• What are the Steps Involved?
• How Fast Can this Happen?

20
The Thermodynamic Hypothesis
The native, folded structure of a protein, under
optimal conditions, is the most energetically
stable conformation possible Christian
Anfinsen, 1972

• Most of the information for determining the
  three-dimensional structure of a protein is
  carried in its amino acid sequence

Anfinsen, C.B. Principles that govern the folding
of protein chains. Science 181, 223-30 (1973).
21
Anfinsens experiments late 1950s through 1960s
Ribonuclease Involved in cleavage of nucleic
acids Structure has a combination of a and b
segments Four disulfide bridges
22
?G, Gibbs Free Energy
Transition state, energy barrier
Reaction Coordinate
23
Entropy and Enthalpy in Protein Folding
Folded Protein
?H, large, negative
?H, small, negative
?S, small, positive
?S, large, positive

• Compensation in entropy and enthalphy for protein
• Contribution of entropy of water molecules
  released upon folding
• ?S of water is large and positive

24
Levinthals Paradox

• Consider a protein of 100 amino acids. Assign
  2 conformations to each amino acid. The total
  conformations of the protein is 21001.27x1030.
  Allow 10-13 sec for the protein to sample through
  one conformation in search for the overall energy
  minimum. The time it needs to sample through all
  conformations is
• (10-13)(1.27x1030)1.27x1017sec 4x109 years!
• Levinthals paradox illustrates that proteins
  must only sample through limited conformations,
  or fold by specific pathways. Much research
  efforts are devoted in searching for the
  principles of the specific pathways.

25
Protein folding

• For any given protein, there is one conformation
  that has significantly lower free energy than any
  other state
• Achieved through kinetic pathway of unstable
  intermediates (not all intermediates are sampled)
• Assisted by chaperones and protein disulfide
  isomerases so intermediates are not trapped in a
  local low energy state

26
The Kinetic Theory of Protein Folding

• Folding proceeds through a definite series of
  steps or a Pathway. A protein does not try out
  all possible rotations of conformational angles,
  but only enough to find the pathway
• - The final state may NOT be the most stable
  conformation possible, but it could be the most
  stable conformation that is accessible in a
  reasonable amount of time. This is also the
  biologically important time frame

27
Folding Pathways

• Protein folding is initiated by reversible and
  rapid formation of local secondary structures
• Secondary structures then form domains through
  the cooperative aggregation of folding nuclei
• Domains finally form the final protein through
  Molten globule intermediates.

28
Models for Protein Folding
1. Hydrophobic collapse. Formation of a 'molten
globule' 2. Framework model. Secondary
structure forms first, perhaps including
supersecondary structure. 3. Nucleation.
Domains fold independently, and sub-domains serve
as 'structural kernels.
29
Kinetics of Protein Folding

• How do proteins fold so fast?
• Currently accepted model is the Pathway Model

• All of the partially folded structures can be
  "funneled" by energy minimizations toward the
  final state.
• Nucleation is critical because it is much more
  difficult to begin an helix than to extend it.

30
Hydrophobic/global collapse

• Alternative model proposed by Ken Dill and
  co-workers (1985s)
• Non-local interactions drive collapse processes
  in proteins and give rise to protein structure,
  stability, and folding kinetics
• Implies that collapse drives secondary structure
  formation rather than the reverse

31
(No Transcript)
32
(No Transcript)
33
Framework model

• Local interactions are the main determinants of
  protein structures
• Interactions as the ones responsible for forming
  secondary structural elements, a-helices and
  b-sheets
• Isolated helices/sheets form early in the protein
  folding pathway, then assemble in the native
  tertiary structure

34
(No Transcript)
35
What's really happening?

• Hydrophobic side chains are being buried
• Secondary structure formation insulates the polar
  protein backbone from the nonpolar protein
  interior
• Hydrogen bonds, disulfide bonds and salt bridges
  begin to form and stabilize structure
• van der Waals interactions bring protein
  substructures into stable contact

36
What's most important in folding?

• Nonspecific interactions (hydrophobic effect, van
  der Waals) are required to bring the protein
  together into a globular conformation
• Steric interactions (restraints on the backbone)
  limit the ways in which the globular conformation
  can form
• Chemically specific interactions (hydrogen bonds,
  ionic interactions, dipolar interactions)
  determine the fine detail of the protein
  structure

37
Folding Funnel Concept
Many Possible Folding Pathways to Get to Native
State
38
Topology determines folding mechanisms

• Protein-folding rates and mechanisms are largely
  determined by a proteins topology rather than
  all its inter-atomic interactions.
• Folding rates of small proteins correlate with
  the average sequence separation between residues
  that make contacts in the 3D-structure, the
  contact order.

Baker, Nature 405, 39 (2000)
39
Contact order
Average separation along the sequence of residues
in physical contact in a folded protein, divided
by the length of the protein
40
Contact Map (2IGD)
Parallel Beta Sheets
Amino Acid Aj
Alpha Helix
Amino Acid Ai
Anti-parallel Beta Sheets
41
Enzymes that speed folding

• Protein disulphide isomerase Facilitates
  formation of correct disulphide bridges
• Peptidyl proline isomerase Catalyses cis-trans
  isomerisation of peptide bonds involving proline
• Molecular chaperones Help folding, especially of
  large proteins, by preventing interaction with
  other proteins

42
Some real time experiments

• temperature jump
• stop - flow
• fluorescence
• NMR
• circular dichroism (CD)

43
Temperature jump

• Background..
• protein is cold (2)
• bang with infra-red laser
• follow with trp fluorescence
• shortest time ?
• about 250 ns
• main information ?
• kinetics
• not much specific structure

44
Stop flow

• Start from chemically unfolded protein
• Use quick mixing / change of conditions to refold
• example
• lysozyme guanadinium HCL
• suddenly dilute in buffer
• protein refolds

45
Stop flow and spectroscopy

• watch with
• circular dichroism (follow secondary structure)
• fluorescence
• (absorption, re-emission, polarisation)
• time scale ?
• gt 10-3 s
• difficulty ?
• fast mixing / dilution

46
NMR

• most detailed structural information
• timescale
• minutes to hours for details
• maybe some seconds for 1-D spectra
• fastest for limited kinds of information
• put in a few labels (19F) peaks can be recorded
  quickly

47
Diseases of Protein Folding

• Prion diseases
• MAD cow (bovine spongiform ecephalopathy, BSE)
• KURU
• Creutzfeld-Jacob
• Fibril or amyloid formation
• Alzheimers
• Parkinsons

48
Prions Protein infectious particles

• Responsible for kuru, Creutzfeld-Jacob disease,
  mad-cow disease, etc.
• The infection involves a change of secondary
  structure and conformation in the prion protein
• A Nobel Prize for Stanley Prusiner in 1997

49
What if protein folding goes wrong ?

• In general misfolded proteins get degraded
  immediately
• In some cases, they can form aggregates, which
  might be difficult to get rid of
• Aggregates can result in severe diseases, such as
  Alzheimers disease and Creutzfeld- Jacob Disease
  (CJD)
• Alzheimers progressive neurodegenerative
  disease characterized by memory loss, impaired
  visuspatial skills, poor judgement etc. Symptoms
  can be easily missed as that due to aging.
• CJD characterized by loss of motor control,
  dementia, paralysis wasting and eventual death.

50
Alzheimers Protein Folding gone Wrong.
An amyloid plaque in Alzheimers disease is a
tangle of protein filaments

• The amyloid protein (42-43 residues) is derived
  by proteolytic cleavage of the amyloid precursor
  protein, a constituent of many healthy cells
• APP has a-helical conformation, while the amyloid
  protein can change into b-conformation forming
  aggregates, and plaques

51
CJD Protein Folding gone Wrong ...
Pathogenic conformation PrPSc
Normal conformation PrPC

• Prion diseases associated with an accumulation,
  in the brain, of prion proteins in the pathogenic
  conformation.
• The normal protein open, greater alpha-helical
  structure, and less beta-sheet structure.
• The pathogenic conformation compact,
  characterized by an increase in the beta-sheet
  structure, relative to that in the normal
  protein.