7. Protein folding, Dynamics & Structural Evolution

protein structures can be determined at atomic resolutions by x-ray crystallography and NMR

the structure is not rigid, there are always fluctuations in the structure over time, i.e proteins are dynamic

the information specifying how a protein folds from an extended chain to its native structure is

contained in its primary a.a sequence

Christian Anfinsen (1957) for ribonuclease a

1. denature RNaseA in 8M urea (abolishes hydrophobic effect) and BME (reduces disulfide bridges)

2. remove urea by dialysis then expose to O2 → 100% enzymatic activity recovered

proteins can follow various folding pathways

forming partially folded conformations with increasing specific structure, until they reach their native state

energy decreases as folding proceeds

complex energy landscape for folding

often proteins do not fold or refold efficently → other proteins can promote folding

1. protein disulfide isomerases: assist in forming correct disulfide bonds

2. proline cis-trans isomerases: accelerate slow interconversion of cis and trans conformations or peptide bonds involving Pro

3. folding chaperones e.g (GroEL-GroES)

GroEL subunits associate to form a dimer of heptamers, hydrophic patches on misfolded proteins bind to hydrophobic surfaces on GroEL

GroES subunits also form a heptamer, which binds to GroEL heptamer after substrate and ATP are bound. this triggers release of misfolded protein into the central cavity, and hydrophobic surfaces of GroEL associate with GRoES

cycles of ATP binding and hydrolysis drive conformational changes in GRoEL/ES according to

1. 7 ATP and 1 misfolded protein bind to a GroEL ring

2. GroES ring binds to the GRoEL ring with bound protein, central cavity further expands, misfolded protein is released into cavity

3. slow ATP hydrolysis allows time for protein folding

4. additional misfolded protein and ATP bind to the other GroEL heptamer

5. binding stimulates release of GroES, ADP and better folded protein

secondary structure prediction

1. 50% accurate when based on physical properties

2. 75% accurate when related (homologous) sequences are also considered

   • limited accuracy b/c tertiary structure and function influence secondary structure

tertiary structure prediction

1. homology modeling: model unknown sequence onto known structure of homolgous protein

2. Ab initio prediction: from chemical principles, and statistics of conformations of stretches of amino acids in known structures

   • e.g Rosetta@home

structural evolution

a common mechanism of protein evolution is duplication of a protein sequence, followed by gradual changes (called divergence) in the primary sequences, this can lead to modified or new structures and functions e.g cytochromes c; myoglobin and hemoglobin

primary sequences of c-type cytochromes are highly divergent but

structure are clearly related (esp. near heme) which is functional site

some sequences can diverge so far that they are

no longer recognizable as related, but the structures are still very similar

some folds, known as superfolds

are particularly tolerant of very different sequences and unrelated funcitons

myoglobin and hemoglobin were the first protein structures to be

solved at atomic resolution using x-ray crystallography (1960, Kendrew & perutz)

Mb and Hb are model systems that illustrate the general principles of

1. structure-function relationship

2. cooperativity

3. allostery

4. mutation and disease

Myoglobin (mb)

• simpler protein found in skeletal and heart muscles

• up to 8% of total protein in muscles of diving animals

• main function in storage of O2 and transport within cells

Hemoglobin (hb)

• more complex structure in red blood cells (RBCs)

• 5 billion RBC/mL of blood, 200 million molecules of Hb/RBC

• transport of O2 from lungs to muscles

• sophisticated delivery system

cooperativity

binding of ligand to one binding site affects the binding of another ligand to another site

myoglobin structure

• single chain of 153 a.a + heme (cofactor)

• chain is ~ 80% helical, forming 8 helices, A-H = globin fold

heme cofactor is a porphyrin or tetrapyrrole

Fe2+ is bound in the centre and is also the site of O2 binding

explain myoglobin structure specifics (3 points)

1. heme is bound between the E and F helices

2. the Fe2+ is coordinated by N of the porphyrin and the N of HIs F8; the 6th coordination site will be occupied by O2

3. His E7 does not coordinate the Fe2+ but restricts the size fo the O2 binding site

Hemoglobin structure

related (homologous) primary sequence and tertiary structure to myoglobin, but consists of 4 subunits: 2 alpha subunits, 2 beta subunits → quaternary structure alpha2beta2

alpha 1 beta 1 and alpha 2 beta 2 form

relatively rigid dimeric units

alpha 1 beta 2, alpha 2 beta 1,

contacts are less extensive and variable

x axis of O2 binding curves

partial pressure of O2

y axis of O2 binding curves

degree of saturation or fractional saturation

dissociation constant Kd for Mb

Kd = [Mb]pO2/[MbO2]

fractionation saturation

number of binding sites/total number of binding sites

yO2 formula for Mb

pO2/pO2 +Kd

p50

the value of pO2 when y = 0.5 i.e half the total binding sites are occupied by O2 which occurs when pO2 = Kd

if pO2 graph shifts right

p50 increases, O2 affinity decreases

if pO2 graph shifts left

p50 decreases, affinity increases

hill plot is used to

assess cooperativity in protein - slope tells us measure of cooperativity

Molecular (perutz) mechanism for Hb cooperativity

hb changes conformation upon O2 binding:

• deoxy Hb, T (tense) state low affinity for O2 → Oxy hb, R (relaxed) state high affinity for O2

size of central cavity in T state Hb

larger central cavity and more salt bridges

R state O2 binding

O2 binds tightly at all 4 binding sites

Hb: binding of 1st O2 is relatively difficult;

after this binding, structure switches from T to R, remaining binding sites relax into high affinity conformation so that subsequent O2s bind more readily: gives sigmoidal binding curve

networks of salt bridges and hydrogen bonds found only in

deoxy Hb - T state

overview of structural changes from T → R

1. electronic effect of binding of O2 to Fe2+ (heme flattens)

2. local rearrangement of His F8

3. quaternary shift that steps alpha 1 C - Beta 2 FG contact one turn along the alpha 1 C helix

4. alpha 10Beta 2and Alpha 2-Beta 1 interfaces msut move simultaneously, due to inflexibility of alpha 1-beta 1 and alpha 2-beta 2 interfaces; breakage of many salt pridges

biological importance of T → R switch

Hb binds O2 in the lungs where pO2 is high (~100 toor) and releases O2 in the body where pO2 is low (~30 torr)

Mb has higher affinity for O2 than Hb, so binds O2 at low pO2

allostery

binding of secondary ligand to a site distinct from primary ligand site influences the binding of primary ligand

Bohr effect

Hb releases H+ upon binding O2 (or other ligands) i.e low pH favours O2 releases

a muscles, hemoglobin preferred

T state - unbound (want to drop off O2 to muscles)

at lungs, hemoglobin prefferred

R state - bound (need to pick up oxygen)

bohr effect: metabolism in active cells generates lactic acid as well as Co2, which reacts with H2O to form H2CO3 (another acid)

causes pH to decrease

bohr effect mechanism

at lower pH formation of salt bridges (formed in T but not R) is favoured due to protonation of alpha chain amino terminus and Beta chain His 146

BPG effect

BPG binds in the central cavity between the subunits in the T state → favours the T state and hence O2 release

BPG

effectively decreases the affinity of Hb ofor O2 so p50 is increased, shifts graph to rght

biological implications of BPG

1. increased BPG levels at high altitude favour more effective release of O2 in body

2. fetal HB has lower BPG affinity than maternal HB, favouring transfer of O2 from maternal HB to fetal HB

sickle cells cause blood flow blockages because

blood cells become distorted due to formation of long fibres of HbS

fibre formation in HbS

favoured by mutation of Glu to Val at Helix A3 position in Beta chain: Val binds to hydrophobic pocket formed by Phe85 to Leu88 between helices E and F

many diseases are caused by mutations of a single amino acid

e.g many protein misfolding diseases, which are often neurodegenerative diseases, cancer, etc