Protein Structure Determination Protein Folding Molecular Chaperones Prions Alzyheimers

Tertiary Structure of Proteins

Two methods: 1. X-RAY diffraction crystal structure 2. NMR solution structure

This is a crystal X-ray diffraction pattern of sperm whale myoglobin

Electron density map

2.0

1.5

1.1

From the diffraction pattern (spots and intensity) one can get a mathematical description of the electron density of a molecule. With proper model construction a 3-D image of the protein is constructed.

NMR

By using chemical shifts of backbone hydrogens and their chemical splitting bond angles can be determined. COSY NMR or Correlated Spectroscopy. By manipulating parameters protons that are close to each other in space but not linked through bonds can be determined by NOSY NMR or Nuclear Overhauser spectroscopy. Growing the protein in bacteria where the carbon source can be substituted by 13C and the nitrogen by 15N (stable isotope substitution) more restraints can be achieved.

The liquid structure(s) can be determined as a group that fit a certain structure space.

Quaternary Structure and Symmetry

Subunits can associate noncovalently, subunits are protomers if identical. Protomer subunits are symmetrically arranged Only rotational symmetry allowed. i.e. cyclic symmetry C2, C3, C6 etc. Dihedral symmetry N-fold intersects a two-fold rotational symmetry at right angles Other higher order types, octahedral or tetrahedral

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.

Physical nature of protein folding

Denatured protein makes many interactions with the solvent water During folding transition exchanges these noncovalent 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 entropy
S = 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 (Cp)
icebergs of solvent around exposed hydrophobics Weakly structured regions in the denatured state

The hydrophobic effect

High Cp changes enthalpy significantly with temperature

For a two state reversible transition

HD-N(T2) = HD-N(T1) + Cp(T2 T1)

As Cp is positive the enthalpy becomes more positive i.e. favors the native state

High Cp changes entropy with temperature

For a two state reversible transition

SD-N(T2) = SD-N(T1) + CpT2 / T1

As Cp is positive the entropy becomes more positive i.e. favors the denatured state

Free energy of unfolding

For GD-N = HD-N - TSD-N Gives
GD-N(T2) = HD-N(T1) + Cp(T2 T1)- T2(SD-N(T1) + CpT2 / T1)

As temperature increases TSD-N increases and causes the protein to unfold

Cold unfolding
Due to the high value of Cp Lowering the temperature lowers the enthalpy decreases Tc = T2m / (Tm + 2(HD-N / Cp) i.e. Tm ~ 2 (HD-N ) / Cp

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 GD-N = HD-N - TSD-N

Solvent denaturation continued

Thus free energy is given by GD-N = GH2OD-N - mD-N [denaturant]

Acid - Base denaturation

Most proteins denature at extremes of pH Primarily due to perturbed pKas 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

Energy profiles during Protein Folding

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. Antiparallel 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)