Levels of Protein Structure

Primary: sequence (translation)

Secondary: backbone H-bonds

Tertiary: Side Chain interactions

Quaternary: 2+ polypeptides

Gene → Sequence → Structure → Function YMGCFTSSGLIVVEHY

Protein folding

Heme Group = Iron (Fe2+) + porphyrin ring

Pictures courtesy of Wikipedia

Heme Group a prosthetic group (cofactor) in myoglobin and hemoglobin

Heme Group a prosthetic group (cofactor) in myoglobin and hemoglobin

Both myoglobin and hemoglobin have two conserved His residues that coordinate the Fe ion in the Heme Group

Proximal (near)Distal

(room for Ligand )deoxyhemoglobin = H2O

oxyhemoglobin = O2

Myoglobin vs. Hemoglobin

single polypeptide a2b2

muscle RBC’sbinds O2 (P50 = 2 torr) binds O2 (P50 = 26 torr)storage/local transport transport

simple binding allosteric – regulation BPG (-)

H+ (-) & CO2 (-)

mito

mito

mito

mito

Hb

Mb

Muscle cell

blood

Hb is an allosteric protein ― One characteristic of this is the sigmoidal shape of the saturation curve.

Would oxygen delivery in the blood be improved if RBCs contained Mb rather than Hb? a) yes b) no c) no difference

Hb is an allosteric protein ― One characteristic of this is the sigmoidal shape of the saturation curve.

In addition Hb as less affinity for O2. Tighter binding of O2 to Hb would be detrimental to the function of the protein.

b HEMOGLOBIN: Rat vs. HumanVHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQRVHLTDAEKAA VNGLWGKVNP DDVGGEALGR LLVVYPWTQR

FFESFGDLST PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN YFDSFGDLSS ASAIM GNPKV KAHGKKHLDN FNDGLKHLDN

LKGTFATLSE LHCDKLHVDP ENFRLLGNVL VCVLAHHFGK LKGTFAHLSE LHCDKLHVDP ENFRLLGNMI VIVLGAHHLGK

EFTPPVQAAY QKVVAGVANA LAHKYHEFTPCAQAAF QKVVAGVASA LAHKYH

CONSERVED RESIDUE: (in green)Amino Acid that is the same in all versions (species) of a protein.

CRITICAL RESIDUE:Amino Acid that is required for the function of the protein. Any replacement will cause protein malfunction.

OO

VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQRVHLTDAEKAA VNGLWGKVNP DDVGGEALGR LLVVYPWTQR

Glycine’s small size allows helix B and helix E to cross.

Proline forces a helix break between helix B and C in Hb.

The amino acids sequence predetermines (to an extent that biochemists can predict with > 80% accuracy) what kind of 2ndary structure a polypeptide segment of a protein will possess.

VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQRVHLTDAEKAA VNGLWGKVNP DDVGGEALGR LLVVYPWTQR

Proline is a unique amino acid in that it has a cyclic side chain. This restricts its F and Y angles to values that are incompatible to a-helix. This is why it ‘breaks’ the a-helix in Hb.

Glycine (a small amino acid) allows two helices in Hb to cross without steric hindrance. Any other side chain in this spot would disrupt the correct functional structure of Hb.

In order to bind O2 (or because O2 binds) the following changes occurIn hemoglobin structure …. 1) salt bridges in the a-b interface are broken 2) there is movement up to 6Å at this interface 3) the 0.5Å Fe moves into the heme plane. 4) the protein structure becomes more ‘relaxed’ 5) The allosteric site for BPG disappears

CH2 – OH |CH – OH |CH2 – OH

Name this molecule. a) glycerol b) 3-propanol c) proprionaldehyde

What do you get if you oxidize an alcohol? a) ester b) amide c) carboxylic acid

COO-

|CH – OH |CH2 – OH

Name this molecule. a) glyceraldehyde b) glyceric acid c) glycerate

Add two phosphate esters to this molecule

COO-

|CH – O – PO3

2-

|CH2 – O – PO3

2-

Name this molecule.

2,3-Bisphosphoglycerate

BPG is the principle allosteric regulator of Hemoglobin.It is produced in RBCs as a ‘bypass’ in Glycolysis.If BPG were not present in RBCs Hb binding to O2 would look like Mb.

ReviewMyoglobin: Muscle cells, P50 = 2 torr unregulated hyperbolic saturation plot (frac. Bound vs. PO2)Hemoglobin: RBCs, P50 = 26 torr – allosteric regulator = 2,3-BPG; sigmoidal saturation plotHb conserved/critical residues G allows 2 helices to cross due to small size P force break in helix B 2H residues from proximal and distal coordination with Fe-hemeStructural changes in Hb deoxy = T (tense) binds BPG, H+, CO2

oxy = R (relaxed) binds O2 cooperatively.

BPG

+ BPG

inactive active

T ↔ R

T-BPG ↔ R-(O2)4

BPG ↕

↕ 4O2

All or none

BPG ↔ ↕ T-BPG

↕R-(O2)4

↔ 4O2

What will happen to [R] when BPG is added? a) ↑ b) ↓ c) ↔

COO- |CH ― OPO3

2-

|CH2 ― OPO3

2-

2,3-BPG is a negative allosteric regulator of Hb It is made in RBCs due to an extra glycolytic enzyme.

Allosteric regulator ― negative ― Binds only to inactive form of protein at allosteric site. positive ― Binds only to active form of protein at allosteric site.

BPG + BPG+ O2

Allosteric regulator ― The conformational difference between deoxyHb and oxyHb involves up to 6Ǻ changes in position of some side chains. In oxyHb the environment that allows deoxyHb to bind BPG is altered and the binding site destroyed.

Hb central cavity

BPG

+His143

+NH3-

+His2

His2+

-NH3+

Lys82+

+His143

b

a

a

b

Salt Bridges and a complementary shape allow BPG to bind to the central cavity in the quaternary structure of hemoglobin.

Hb is an allosteric protein ― Without BPG present the O2 saturation plot for Hb would appear the same as the O2 saturation plot for Mb.Although technically a “feedback” (-) allosteric regulator the [BPG] in RBCs is essentially constant and therefore is not actually regulating in response to changes in environment. H+ and CO2 are the real-time “feedback” regulators.

oxyHis 146 pK = 6.5 (basic)

89%

― Asp 94 deoxy His 146 +

pK = 8.0 (acidic) 80%

The Bohr Effect ― Hb has a lower affinity for O2 as pH↓ or CO2↑.

The added stability of the H146-D94 salt bridge found only in deoxyHb is caused when H146 is protonated. A lower pH induces the conformational change.

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

-9 -7 -5 -3 -1 1 3 5 7 9

HbF = a2g2 HbA = a2b2

Fetal Hb ― HbF a2g2 is expressed by a fetus instead of HbA (a2b2).

HbF has a higher affinity for O2 than HbA. This is due to the lower affinity of HbF for 2,3-BPG which is due to the mutation b-His-143 → Ser.

Hb central cavity

BPG

+His143

+NH3-

+His2

His2+

-NH3+

Lys82+

+His143

b

a

a

bIn fetal Hemoglobin the mutation of H143S weakens the binding of the (-) regulator BPG. This strengthens the binding to O2.

Ser143 g

g Ser143

Fetal Hemoblobin

HbS ― Sickle Cell Hb is caused by the mutation b-Glu-6 → Val. The nonpolar Val is exposed and causes the aggregation of deoxyHb. This influences the shape of the RBCs. The sickle cells are more readily lysed in the blood leading to anemia.

Sickle cells, however, are resistant to the parasite that causes malaria.

Sickle cell diseaseHomozygous normal

Heterozygous asymptomatic carrier

Individuals who are heterozygous for the sickle trait have a selective advantage over homozygous individuals where malaria is endemic.

For effective transport affinity must vary with pO2