Lectures 5-6 - GHOSE LAB · Lecture 5-6: Outline Chapter 5: Protein Function 2 • Terms/concepts...

Based on Profs. Kevin Gardner & Reza Khayat 1

Biochemistry - I

Mondays and Wednesdays 9:30-10:45 AM (MR-1307)

SPRING 2017

Lectures 5-6

Lecture 5-6: Outline

2Chapter 5: Protein Function

• Terms/concepts • The vertebrate oxygen transport and storage system • The oxygen binding site of myoglobin (Mb) • Oxygen binding and release by myoglobin • Oxygen transport by hemoglobin (Hb) • Effects of other ligands on Hb • Hemoglobin-based disease

• not covered in class, read text: Immunoglobulins (Section 5.2) • not covered in class, not on test: Actin, myosin (Section 5.3)

Need for Oxygen Transport• O2 is fairly abundant in air:

– ~21% of air by volume = 210 mL O2/liter of air

• Problem: O2 is only sparsely soluble in liquid: – ~0.5% soluble in water @ 35°C = 5 mL/liter – Insufficient to support more than ~1% of metabolic

processes for typical mammal – O2 also diffuses short distances - about 100 µm

• Solution: O2-carrying system - vasculature, specialized cells and oxygen-carrying proteins to boost O2 solubility – in humans: 150g hemoglobin/liter of blood – Raises O2 solubility to ~20%


Useful Terms & Concepts

4

• Proteins breathe and are dynamic • Ligand is a molecule (e.g. protein, DNA, peptide, hormone…) that binds to

another molecule • Ligands can regulate the activity of a protein (e.g. ON/OFF) • Binding can be reversible or irreversible • Binding site is usually complementary to ligand, with compatible shapes,

charges, etc. to facilitate favorable non-covalent interactions • The binding site may form through an induced fit (conformational change) of

protein and/or ligand • Multi-subunit proteins are composed of multiple polypeptide chains • Conformational change in one subunit of a multisubunit proteins may affect

the conformation of another subunit to regulate the function of the protein (regulation could be positive or negative)

• A ligand that is chemically modified by a protein (i.e. an enzyme) is called a substrate.

• Drugs are ligands/substrates that can reversibly or irreversibly bind to proteins

Chapter 5: Protein Function

The Vertebrate Oxygen Transport and Storage System

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• Animals require a steady supply of O2 to their cells and a steady removal of waste products such as CO2 and CO

• With the exception of insects, diffusion of O2 through blood is not fast enough; therefore nearly all animals pump O2 from their lungs/gills through their arteries to the tissues and return CO2 via venous blood to their lungs/gills

Red blood cells ~6-8µm No organelles(!) and full of hemoglobin


Heme-containing Proteins: O2 Storage and Transport

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• Myoglobin is composed of a single 153 residue polypeptide chain liganded to a prosthetic group called a heme

• Hemoglobin is a hetero-tetramer protein composed of two copies of an α- and two copies of a β-polypeptide chain – two gene products that are homologs (evolutionary related). 4 proteins chains, each of which bind heme

For O2 storage and transport, mammals use: • storage = myoglobin (Mb): stores oxygen in tissues like muscle where there is a high

demand for oxygen • transport = hemoglobin (Hb): transports oxygen from lungs/gills to tissues, and

transports carbon dioxide and protons back to the lungs/gills

Hemes


Heme Structure

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• Composed of two parts: a porphyrin ring (protoporphyrin IX) + iron • Synthesized in mitochondria and cytosol of immature red blood cells • Iron (Fe2+) binds O2 reversibly, Fe3+ does not bind O2. (superoxide = O2

- bound to Fe3+)

• Mb/Hb prevent oxidation of Fe2+ (ferrous) to Fe3+ (ferric) by bound O2.


Mb: Oxygen Storage

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• 16.7 kDa monomer • 8 α-helical segments, ~80% of AA

are in helices • Helices named A, B, C … H; non-

helical connecting bends (AB, etc.)

• Stores O2, mostly in muscle • High concentration in diving

mammals • First protein to have its 3D

structure determined, by X-ray diffraction


Role of Proximal, Distal His in Mb

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proximal Directly-bonded to Fe

distal


Mb: O2 Binding Site

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• Fe(II) is normally octahedrally coordinated –attached to six ligands/binding groups • Nitrogen atoms of porphyrin ring account for 4 ligands • His93 residue of hemoglobin/myoglobin accounts for 1 ligand –the proximal His • O2 accounts for last ligand. This O2 interacts with a His64 residue –the distal His • Carbon monoxide has a stronger affinity for the heme; hence, it is toxic

•Venous blood (purple) [Fe2+] •Arterial blood (bright red) [Fe2+- O2]

•Fe3+ = methemoglobin (ferric Hb) brown (old meat)proximal histidine:

normally bound Fe

distal histidine: does not normally bind Fe


Mb: O2 Specificity & Release

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Gas binding/release: Proteins are dynamic entities –they breathe

• Myoglobin’s breathing opens a path for O2 to escape

• O2 bounces around inside the heme pocket, binding and releasing the heme

• O2 eventually escapes from the binding site

Specificity: Carbon monoxide binds to free heme with 20,000 times higher affinity than does O2, but it binds myoglobin with only 200 times higher affinity than does O2.


Review of Protein/ligand Binding Equilibria

12

Keq = Ka = [PL]/[P][L] Ka = association constant

1/Ka = Kd =[P][L]/[PL] Kd= dissociation constant

Kd is [L] at which half of the available ligand-binding sites are occupied theta (θ) is the fraction of binding sites = binding sites occupied / total binding sites

θ = [PL] / ([PL] + [P]) (goes from 0 to 1)

⬇ Kd = ⬆ affinity

The binding of O2 to myoglobin can be described by the following equilibrium

Kd =[P][L]/[PL] [PL] = [P][L]/Kd Plugin [PL] into theta equation θ = [L]/([L]+Kd) at [L] = Kd, θ = 0.5

P: protein (myoglobin) L: ligand (O2)P + L ⇌ PL

[L] is for the unbound ligand

[L] >> [P] Chapter 5: Protein Function

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Important reminders: • Kd defines the

concentration of free ligand that will result in 50% saturation of ligand

• = more than 50% of the protein is bound to ligand at ligand concentrations > Kd


Mb: Thinking Quantitatively about O2 Binding

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• Myoglobin (muscle) accepts oxygen released from hemoglobin, and delivers the oxygen to the mitochondria when oxygen needs are sufficiently great (e.g. during exercise)

• pO2 = 100 mm Hg in lungs/gills (Hb present) • pO2 = 30 mm Hg in tissues (Mb present)

• The binding of oxygen to myoglobin can be studied using spectroscopy

•X-axis: Increasing concentrations of O2 in buffer (PO2) •Y-axis: Fraction of O2 bound myoglobin

• The concentration of O2 that results in 50% (half saturation) of myoglobin-O2 is called P50. This is the dissociation affinity and is the inverse of the binding affinity of myoglobin for O2

• The strong affinity of myoglobin for O2 is needed in order for myoglobin to successfully bind the limited amount of O2 present in the capillaries ~30 mm Hg

• Myoglobin releases its O2 when the pO2 concentration of the cell drops below myoglobin’s affinity for O2


Hb: Oxygen Transport

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• Best thought of as a dimer of dimers

• Strongest interactions between like-numbered subunits α1-β1 and α2-β2 (circled)

• Weaker interactions between α2-β1 and α1-β2

Each polypeptide chain (α1, α2, β1 and β2) has a heme and binds to O2 = 0-4 O2 bound per Hb

Oxygen binding near α1-β2 and α2-β1, which triggers changes the interface between α1-β2

• Hemoglobin is a heterotetramer: two α-chains (141aa) and two β-chains (146aa)


O2-triggered 4° Structure Changes in Hb

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• Hb exists in two states: • T (tense) state where O2 is absent • R (relaxed) state where O2 is present

• Binding of O2 causes a conformational change in Hb from T- to R- state • Change occurs because ionic interactions between the α2-β1 and α1-β2 are eliminated • The T- to R- transition results in closing of hole between β subunits

Deoxyhemoglobin OxyhemoglobinChapter 5: Protein Function

Deoxy-Hb: T State More Stable

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• Deoxyhemoglobin favors the tense (T) state, which contains a greater number of ion pairs • Oxyhemoglobin favors the relaxed (R) state • Mechanism for this shift: Binding of O2 to heme creates steric clash that changes the α2-β1 and α1-β2 interfaces, shifting Hb from T to R state

• T state binds O2 more weakly than R state • Binding of O2 to one chain shifts T->R, increasing Hb’s affinity for more O2 = positive

cooperativity

Imidazole COO-COO-ε amino

α2β1


Effect of O2 Binding to Hb Heme

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Heme more planar with O2 bound.

Side-chains rotate

Very small changes!


Allostery: Structural Effects of O2 Binding Culminate in Large-scale Changes

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Binding of O2 results in a large number of movements in Hb. These involve movements in the side-chains that are propagated into quaternary movements of the subunits

All protein quaternary changes stem from small changes that propagate and expand into large scale changes


Comparing Mb & Hb O2 binding curves

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(note that Y and θ are same value = fractional saturation)


PO2 = 100 mm Hg in lungs PO2 = 30 mm Hg in tissues

Hemoglobin must bind to O2 in the lungs and release it in the capillaries near tissue

O2 Transport by Hb: Critical to be Positively Cooperative

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Cooperative binding and allostery (hyperbolic vs. sigmoidal binding)

θ

pO2 (mm Hg)

real Hb

“non-cooperative Hb” same p50, but 4

hemes independent

T4

R4

Hb


Why is Hb Positive Co-operativity Important?


Symmetry (Monod/Wyman/Changeux) model:

T state

R state2. O2 binds more tightly to R state

than T state

1. Subunits change conformation in a concerted manner

3. Progressive addition of O2 pushes

T<->R equilibrium towards R

Works well for hemoglobin if α, β considered to be identical; generality perhaps somewhat questionable

Further Regulation and Alteration of Hb Function

• Ligand regulation of Hb: • pH • BPG

• Developmental changes in Hb: embryonic, fetal hemoglobin

• Alteration of Hb function by amino acid substitution


pH Dependence of Oxygen Binding: Bohr Effect

25

• Conformational changes in Hb upon O2 binding leads to the release of ~0.6 protons (H+)

• Le Chatlier’s principle suggests that this will become progressively more unfavorable with lower pH (= higher [H+])

• Hence, lower pH makes it more difficult for O2 to bind and shifts the saturation curve to progressively higher pO2 levels

Interlinking of Bohr effect and CO2 transport

• CO2 transport from periphery is handled:

– ~8% physically dissolved CO2 gas – ~25% carried by Hb via reversible reactions w/amino groups – ~67% transported as bicarbonate

• CO2(g) modestly soluble in H2O; HCO3- highly soluble

• RBC carry carbonic anhydrase to catalyze following conversion:

• In periphery: – Proton produced by this reaction helps facilitate O2 release by Hb

– Hb uptake of H+ helps facilitate carbonic anhydrase

• In lungs – Reverse above process

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CO2 + H2O H+ + HCO3-


Bisphosphoglycerate (BPG)

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• BPG binds tightly to deoxyhemoglobin in 1 BPG:1Hb ratio • Binds poorly to oxyhemoglobin

• Effect: BPG stabilizes deoxy-state, shifting O2 saturation curve to higher pO2

Hb without BPG ~ Mb


Role of BPG in High Altitude Adaptation

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increased BPG levels : shift pO2 to right

Facilitate greater O2 unloading in capillaries

Strategy fails when [BPG] high enough to begin affecting loading in lungs - related to max habitable altitude at 18,000ft


Universal Equation for Hb

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HbT(H+ • BPG • CO2) + O2

HbR(O2) + H+ + BPG + CO2

NOT meant to infer stoichiometry


CO/CN Poisoning

• CO binds Fe(II) in Hb-bound heme ~ 200x stronger than O2, hence can block O2 transport

• As addressed last lecture, CO binds Fe(II) in free heme even more tightly than in Hb-bound – Distal histidine lowers CO affinity by preventing direct linear

interaction between CO, Fe(II)

• CN poisoning due to interactions with Fe(III) in proteins other than Hb; clinical treatment for CN poisoning involves intentionally generating Fe(III)-Hb to soak up excess CN

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Developmental Regulation of Hb Isoforms

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Adult Hb (HbA): α2β2 >> α2δ2

Embryonic Hb: ζ2ε2

Fetal Hb (HbF): α2γ2

Need for Developmental Regulation: Obtaining O2 from Maternal Hb

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Fetal Hb must have higher oxygen affinity than maternal in order to ensure that the fetus can obtain O2 from maternal blood via transfer at the placenta

This higher O2 affinity is achieved by the use of isoforms of Hb (HbF) that have lower affinity for BPG

Lower affinity for BPG = higher affinity for O2

HbS: Sickle Cell Anemia• Sickle cell anemia originates from HbS, a form that contains a mutation

replacing Glu 6 with a Val residue on the β-chains (E6V)

• This mutation generates a hydrophobic patch on the surface of Hb that is exposed only when Hb is deoxygenated. Such patches can lead to interactions between different Hb complexes, eventually forming long fibers that distort the shape of RBC into sickled appearance

– The kinetics of process, like many aggregation-type processes, are highly non-linear with respect to monomer concentration. In this case, the rate is dependent on [HbS]10

– Heterozygotes in HbS are asymptomatic as their [HbS] is half that of homozygotes; 0.510 ~ 0.001

• Fiber formation often occurs in small blood vessels, blocking these vessels and setting up vicious cycle


HbS: E6V Mutation in Fibers

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E6V mutation


Hb Molecular Pathology• Over 500 variant Hb forms have been discovered to date, 95%

of which contain single (“point”) mutations

• Identified by electrophoresis, especially IEF

• Classes of mutations: – Surface: alter protein solubility (>50% of all mutations known;

conservation pattern) – Internal: destabilize Hb, cause problems by way of high levels of heme

degradation products – Stabilize Fe(III)-Hb (methemoglobin): generate permanent Fe ligand from

sidechain in close proximity – Alter α1β2 interface: prevent changes in 4° structure that should

accompany O2 binding. Often manifests as reduced co-operativity (nH ~ 1)


• α-, β-thalassemias named for chain with defective production; remaining material has pathological properties

• May arise from mutation in coding sequence of hemoglobin (=hemoglobinopathy), but not necessarily

• Many occur from deletion of one of the Hb genes:

Thalassemias: Hb Production Deficiencies


Importance of Surface Charge: Evolution of Mb

37

Higher charge (ZMb) on myoglobins from

vertebrates with increased max

concentrations in muscle


Lectures 5-6 - GHOSE LAB · Lecture 5-6: Outline Chapter 5: Protein Function 2 • Terms/concepts...

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