Enzyme Kinetics & Protein Folding

9/7/2004

Protein folding is “one of the great unsolved problems of science”

Alan Fersht

protein folding can be seen as a connection between the genome (sequence) and what the

proteins actually do (their function).

Protein folding problem

• Prediction of three dimensional structure from its amino acid sequence

• Translate “Linear” DNA Sequence data to spatial information

Why solve the folding problem?

• Acquisition of sequence data relatively quick• Acquisition of experimental structural information

slow• Limited to proteins that crystallize or stable in

solution for NMR

Protein folding dynamics

Electrostatics, hydrogen bonds and van der Waals forces hold a protein together.

Hydrophobic effects force global protein conformation.

Peptide chains can be cross-linked by disulfides, Zinc, heme or other liganding compounds. Zinc has a complete d orbital , one stable oxidation state and forms ligands with sulfur, nitrogen and oxygen.

Proteins refold very rapidly and generally in only one stable conformation.

The sequence contains all the information to specify 3-D structure

Random search and the Levinthal paradox

• The initial stages of folding must be nearly random, but if the entire process was a random search it would require too much time. Consider a 100 residue protein. If each residue is considered to have just 3 possible conformations the total number of conformations of the protein is 3100. Conformational changes occur on a time scale of 10-13 seconds i.e. the time required to sample all possible conformations would be 3100 x 10-13 seconds which is about 1027 years. Even if a significant proportion of these conformations are sterically disallowed the folding time would still be astronomical. Proteins are known to fold on a time scale of seconds to minutes and hence energy barriers probably cause the protein to fold along a definite pathway.

Energy profiles during Protein Folding

Physical nature of protein folding

• Denatured protein makes many interactions with the solvent water

• During folding transition exchanges these non-covalent interactions with others it makes with itself

What happens if proteins don't fold correctly?

• Diseases such as Alzheimer's disease, cystic fibrosis, Mad Cow disease, an inherited form of emphysema, and even many cancers are believed to result from protein misfolding

Protein folding is a balance of forces

• Proteins are only marginally stable • Free energies of unfolding ~5-15 kcal/mol• The protein fold depends on the summation of all

interaction energies between any two individual atoms in the native state

• Also depends on interactions that individual atoms make with water in the denatured state

Protein denaturation

• Can be denatured depending on chemical environment– Heat

– Chemical denaturant

– pH

– High pressure

Thermodynamics of unfolding

• Denatured state has a high configurational entropyS = k ln W

Where W is the number of accessible states

K is the Boltzmann constant

• Native state confirmationally restricted• Loss of entropy balanced by a gain in enthalpy

Entropy and enthaply of water must be added

• The contribution of water has two important consequences– Entropy of release of water upon folding

– The specific heat of unfolding (ΔCp)• “icebergs” of solvent around exposed hydrophobics

• Weakly structured regions in the denatured state

The hydrophobic effect

High ΔCp changes enthalpy significantly with temperature

• For a two state reversible transition

ΔHD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1)

• As ΔCp is positive the enthalpy becomes more positive

• i.e. favors the native state

High ΔCp changes entropy with temperature

• For a two state reversible transition

ΔSD-N(T2) = ΔSD-N(T1) + ΔCpT2 / T1

• As ΔCp is positive the entropy becomes more positive

• i.e. favors the denatured state

Free energy of unfolding

• For

ΔGD-N = ΔHD-N - TΔSD-N

• GivesΔGD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1)- T2(ΔSD-N(T1) + ΔCpT2 / T1)

• As temperature increases TΔSD-N increases and causes the

protein to unfold

Cold unfolding

• Due to the high value of ΔCp

• Lowering the temperature lowers the enthalpy decreases

Tc = T2m / (Tm + 2(ΔHD-N / ΔCp)

i.e. Tm ~ 2 (ΔHD-N ) / ΔCp

Measuring thermal denaturation

Solvent denaturation

• Guanidinium chloride (GdmCl) H2N+=C(NH2)2.Cl-

• Urea H2NCONH2

• Solublize all constitutive parts of a protein

• Free energy transfer from water to denaturant solutions is linearly dependent on the concentration of the denaturant

• Thus free energy is given by

ΔGD-N = ΔHD-N - TΔSD-N

Solvent denaturation continued

• Thus free energy is given by

ΔGD-N = ΔGH2OD-N - mD-N [denaturant]

Acid - Base denaturation

• Most protein’s denature at extremes of pH

• Primarily due to perturbed pKa’s of buried groups

• e.g. buried salt bridges

Two state transitions

• Proteins have a folded (N) and unfolded (D) state

• May have an intermediate state (I)

• Many proteins undergo a simple two state transition

D <—> N

Folding of a 20-mer poly Ala

Unfolding of the DNA Binding Domain of HIV Integrase

Two state transitions in multi-state reactions

Rate determining steps

Theories of protein folding

• N-terminal folding

• Hydrophobic collapse

• The framework model

• Directed folding

• Proline cis-trans isomerisation

• Nucleation condensation

Molecular Chaperones

• Three dimensional structure encoded in sequence• in vivo versus in vitro folding• Many obstacles to folding

D<---->N

Ag

Molecular Chaperone Function

• Disulfide isomerases• Peptidyl-prolyl isomerases (cyclophilin, FK506)• Bind the denatured state formed on ribozome• Heat shock proteins Hsp (DnaK)• Protein export & delivery SecB

What happens if proteins don't fold correctly?

• Diseases such as Alzheimer's disease, cystic fibrosis, Mad Cow disease, an inherited form of emphysema, and even many cancers are believed to result from protein misfolding

GroEL

GroEL (HSP60 Cpn60)

• Member of the Hsp60 class of chaperones• Essential for growth of E. Coli cells• Successful folding coupled in vivo to ATP

hydrolysis• Some substrates work without ATP in vitro• 14 identical subunits each 57 kDa• Forms a cylinder• Binds GroES

GroEL is allosteric

• Weak and tight binding states

• Undergoes a series of conformation changes upon binding ligands

• Hydrolysis of ATP follows classic sigmoidal kinetics

Sigmoidal Kinetics

• Positive cooperativity• Multiple binding sites

Allosteric nature of GroEL

GroEL changes affinity for denatured proteins

• GroEL binds tightly

• GroEL/GroES complex much more weakly

GroEL has unfolding activity

• Annealing mechanism

• Every time the unfolded state reacts it partitions to give a proportion kfold/(kmisfold + Kfold) of correctly folded state

• Successive rounds of annealing and refolding decrease the amount of misfolded product

GroEL slows down individual steps in folding

• GroEL14 slows barnase refolding 400 X slower

• GroEL14/GroES7 complex slows barnase refolding 4 fold

• Truncation of hydrophobic sidechains leads to weaker binding and less retardation of folding

Active site of GroEL

• Residues 191-345 form a mini chaperone

• Flexible hydrophobic patch

Role of ATP hydrolysis

The GroEL Cycle

A real folding funnel

Amyloids

• A last type of effect of misfolded protein

• protein deposits in the cells as fibrils

• A number of common diseases of old age, such as Alzheimer's disease fit into this category, and in some cases an inherited version occurs, which has enabled study of the defective protein

Known amyloidogenic peptides

CJD  spongiform encepalopathies  prion protein fragments  APP  Alzheimer  beta protein fragment 1-40/43 HRA  hemodialysis-related amyloidosis  beta-2 microglobin* PSA  primary systmatic amyloidosis  immunoglobulin light chain and fragments SAA 1  secondary systmatic amyloidosis  serum amyloid A 78 residue fragment FAP I**  familial amyloid polyneuropathy I  transthyretin fragments, 50+ allels FAP III  familial amyloid polyneuropathy III  apolipoprotein A-1 fragments CAA  cerebral amyloid angiopathy  cystatin C minus 10 residues FHSA  Finnish hereditary systemic amyloidosis  gelsolin 71 aa fragment IAPP  type II diabetes  islet amyloid polypeptide fragment (amylin) ILA  injection-localized amyloidosis  insulin CAL  medullary thyroid carcinoma  calcitonin fragments ANF  atrial amyloidosis  atrial natriuretic factor NNSA  non-neuropathic systemic amylodosis  lysozyme and fragments HRA  hereditary renal amyloidosis  fibrinogen fragments

Transthyretin

• transports thyroxin and retinol binding protein in the bloodstream and cerebrospinal fluid

• senile systemic amyloidosis, which affects  people over 80, transtherytin forms fibrillar deposits in the heart. which leads to congestive heart failure

• Familial amyloid polyneuropathy (FAP) affects much younger people; causing protein deposits in the heart, and in many other tissues; deposits around nerves can lead to paralysis

Transthyretin structure• tetrameric. Each monomer has two 4-stranded-sheets, and a short -helix. Anti-parallel beta-sheet interactions link monomers into dimers and a short loop from each monomer forms the main dimer-dimer interaction. These pairs of loops keep the two halves of the structure apart forming an internal channel.

Fibril structure

• Study of the fibrils is difficult because of its insolubility making NMR solution studies impossible and they do not make good crystals

• X-ray diffraction, indicates a pattern consistent with a long -helical structure, with 24 -strands per turn of the -helix.

Formation of proto-filaments

• Four twisted -helices make up a proto-filament (50-60A)

• Four of these associate to form a fibril as seen in electron microscopy (130A)