Folding and Flexibility

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Folding and Flexibility
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Globular proteins Marginally stable structures
The unfolded state is extended and flexible. The
folded state is globular and compact.

• Conversion from native to denatured state facile.
  Weak forces at play, can be just 5-15 kcal/mol
  (H bond 2-5 kcal/mol)
• Enthalpy energy of non-covalent bonds
  hydrophobic interaction, H bonds, ionic bonds.
  Although covalent bonds contribute to enthalpy,
  they do not change from native to denatured state
  (except for possible disulfides).
• Enthalpy interactions more favorable in packed
  native state rather than unpacked denatured. Can
  be up to several hundred kcal/mol
• Entropy denatured state highly disordered, more
  favorable. Energy difference can also be several
  hundred kcal/mol.
• Total free energy difference between native and
  denatured is difference of enthalpy and entropy.
  This small difference complicates ability to
  predict possible native states.

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Denaturation of proteins

• Proteins unfold and lose secondary structure from
  a variety of agents.
• Can be due to heat, chaotropic salts, pH etc.
• Variety of signals can be followed to monitored
  denaturation. Intrinsic fluorescence of Rnase A
  or circular dichroism (measure amount of helical
  structure).
• Abrupt increase from nature to denatured state
  suggest that the process is cooperative.

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Refolding of RNAse A

• RNAse A is special case. Enzyme extremely
  stable.
• Experiments done by Anfinsen in 1950s.
• Urea is chaotropic salt and causes denaturation.
  
• Mercaptoethanol reduces disulfide bonds.
• The unfolded state is inactive.
• Removal of urea and mercaptoethanol resulted in
  refolding to yield active enzyme.
• What would happen if mercaptoethanol was removed
  first and then the urea?
• What these experiments showed was that tertiary
  structure determined by primary structure.

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Kinetics of refolding

• X-ray structure of several hundred proteins show
  that specific sequence of polypeptide has the
  same fold.
• Are all conformations sampled in random fashion
  until lowest energy form is found?
• Levinthal showed this cant be. If each peptide
  group has 3 different conformations (a, b, L of
  Ramachandran plot) and could interconvert in pico
  (10-12) sec, a peptide of 150 residues would have
  3150 possible conformations and it would take
  1048 yr to fold properly. Actually time is 0.1
  to 1000 sec. Thus kinetic pathway exist which
  prevents sampling irrelevant conformations.

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Refolding is stepwise and hierarchical.

• Local secondary structure forms first and this is
  followed by longer range interactions

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Molten globule state an intermediate

• First observable folding event with some proteins
  is collapse of of flexible disordered polypeptide
  into partially organized globular state, called
  molten globule. This is fast, within deadtime of
  stop flows

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Molten globule state contd

• Molten globule has most of the secondary
  structure of native protein.
• It even has correct positions of a-helix and
  b-sheets. Less compact than native protein.
  Proper packing of interior has not yet occurred.
  Internal side chains more flexible than in
  native.
• Loops and other surface components not in correct
  conformation.
• View as ensemble of related structures
• Next step is slow and can be up to 1 sec. Native
  like elements of tertiary structure develop and
  form subdomains that are not fully developed.
  Still far from single form, still ensemble.
• Final stage involves native interactions,
  hydrophobic packing, and fixation of surface loops

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Free energy funnel

• Unfolded is high degree of conformational entropy
  and high energy.
• As folding progresses, narrowing of funnel
  represents decrease in number of conformational
  species.
• Small depressions along side are semistable
  intermediates that can slow down folding process.
• At bottom, ensemble of folding intermediates
  reduced to single native conformation.

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Burying hydrophobic side chains

• Energy gain from H bonds of secondary structure
  is not a significant driving force. There are
  also H bonds in unfolded state with water.
• Large free energy change by bringing hydrophobic
  side chains out of water (increase entropy).
• Packing residues into hydrophobic core minimizes
  number of conformation (steric consideration).
• NH and CO groups no longer can interact with
  water in interior, thus H bonding among
  themselves provides more favorable energetics
  thus a-helix and b-sheet are formed.
• Big factor thus is burying hydrophobic residues

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Thermodynamics of refolding

• Folding dependent on the Gibbs free energy
  difference between folded and unfolded state
• DGoverall DGF - DGU DH - TDS
• (HF - HU) T(SF SU)
• In unfolded state, peptide chain and side chains
  interact with water, thus energetics must also
  account for water.
• DGtotal DHchain DHsolvent - TDSchain -
  DSsolvent

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• Folded protein is highly structured, DS of
  unfolded to folded is negative.
• The DH depends on R group. For nonpolar
  residues, folded state yields favorable van der
  Waal interactions (weak). In unfolded,
  interaction with water yields induced dipoles.
  This produces electrostatic interactions. For
  water, the DH for nonpolar groups is negative in
  favor of folded state because folding allows
  water to have favorable interactions with itself
  rather than with nonpolar side chain. The DH is
  small for nonpolar side chains.
• The DSsolvent for nonpolar groups is large
  positive and favors folded state. This is due to
  order forced upon water by hydrophobic groups
• For polar side chains, the DHchain is positive
  and the DHsolvent is negative. Because solvent
  molecules are somewhat ordered even around polar
  groups, the DSsolvent is small and positive.
• Most significant factor is the DSsolvent

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Enzymes assist in disulfide bond formation

• Disulfide bonds formed only in extracellular
  proteins (or those in periplasmic space). In
  bacteria, catalyzed by disulfide bridge forming
  enzyme (Dsb). In eukaryotes, formed by protein
  disulfide isomerase, PDI of e.r.
• Studies on bovine pancreatic trypsin inhibitor
  BPTI show importance of disulfides. Protein has
  6 Cys, 3 disulfide and has 58 residues.

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BPTI folding pathway

• Fully reduced BPTI is unfolded and does not fold
  until Cys residues oxidized to disulfide.

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Proline isomerization

• The continuation of the peptide backbone coming
  off the peptide bond can be cis or trans. Cis is
  1000 less stable than trans form.
• In proline, cis is only around 4 times less
  stable than trans. Cis is found at certain
  prolines at bends. In native structure, these
  cis forms are stabilized by energy of tertiary
  structure. In unfolded state, energy constraints
  gone and there is equilibrium between cis and
  trans. Isomerization is slow and is
  rate-limiting in vitro for protein folding.

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Chaperonins

• As proteins are being translated, hydrophobic
  patches can be exposed to solvent. This can
  cause aggregation and incorrect folding.
• Molecular chaperones prevent aggregation by
  binding with hydrophobic patches
• First discovered as heat shock proteins (Hsp 70),
  protein unfolding increased at high temp. Also
  expressed constitutively.
• Hsp70 has ATP binding region and a second region
  that binds hydrophobic regions. No structural
  data on the complete molecule
• E. coli chaperones GroEL (Hsp 60) and GroES (Hsp
  10) well characterized. Together, they function
  as complex called chaperonin

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GroEL

• Assembles as 14 subunits, forms two rings where
  each ring has 7 subunits.
• Protein has 3 domains, apical, intermediate, and
  equatorial. Equatorial is largest, mainly
  a-helical. It plays key role. Provides contacts
  between each subunit and also between rings.
  Also is binding site for ATP.

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GroEL and GroES complex

• Each complex has two large pockets formed from
  two heptameric rings (red and in blue).
• GroES is also a heptameric ring (yellow).

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Mode of action of GroEL-GroES complex