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Chapter 7 - Protein Function

Important Principles

• Key features of protein function demonstratedby O2-binding proteins (Hb, Mb), Immunesystem proteins and Muscle proteins

• Proteins are flexible, not rigid

• Protein function often depends on theirinteraction with ligands

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• Myoglobin & Hemoglobin contain a hemeprosthetic group that binds 02 reversibly

• Hemoglobin is a multi-subunit protein that

binds 02 cooperatively

• Hb binds H+, CO2 and 2,3 bisphosphoglyceratein a manner that affects the affinity for 02

• Sickle-cell anemia illustrates the importance ofconformation to function

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• The immune system utilizes extremely specificinteractions between proteins and foreignmolecules to protect the organism

• Protein flexibility and interactions areillustrated by the cyclic conformationalchanges that cause muscles to contract

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Protein function

• Protein function almost always depends oninteractions with other molecules (ligands)– H+, O2, ATP, lipids, peptides, proteins, RNA, etc.

• Transient nature of protein-ligand interactions(noncovalent) allows for flexibility in response

• Ligands bind at binding sites– Complementary in shape, size, charge,

hydrophobic or hydrophilic character of ligand

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• Protein-ligand interac tion is specific - veryselective binding

• proteins may have separate binding sites formultiple ligands

• Proteins are flexible - subtle or dramaticchanges in structure to accomodate ligands

• Binding coupled to conformational changes -“induced fit”

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• In multimeric proteins, conformationalchanges often “contagious”

• Special cases of Enzymes– Ligands = substrates– Binding site = catalytic site or “active site”

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Reversible binding of ligands - 02 proteins

• Myoglobin (Mb) & Hemoglobin (Hb)– Most studied, best understood– Illustrate most aspects of ligand binding

Mb

Fig 7.3

Hb

Fig 6.23a

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Oxygen binding proteins (Mb & Hb)

Proteins evolved to store & transport gases

– O2 poorly soluble

– Diffusion through tissues low

O2 bound to Heme Prosthetic group

– No αα side chain suited for binding of O2

– Fe & Cu bind O2, but form free radicals

– O2-binding properties of Fe exploited bysequestering it in less reactive form

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Heme - organic ring structure whichbinds single Fe2+

Sequestering heme prevents irreversibleoxidation of Fe2+ to Fe3+

Fig 7.1a Fig . 7.1b

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Myoglobin (Mb) - Single O2 binding protein

• Mr = 16,700

• Found in almost allmammals

• Primarily muscle tissue

• particularly abundantin diving mammals

• 153 αα’s; 1 hememolecule

• Member of globinfamily of proteins

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Protein-ligand interac tions can be describedquantitatively

• Mb function depends on ability to bind/release

O2 where needed

• Quantitative description central to

understanding

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Equations of the form x=__y�__ describe hyperbolas y + z

[L] at which 1/2 of the available binding sitesare occupied (θ = 0.5) = 1/Ka=Kd

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For Mb, Kd = [O2] ([L]) at which 1/2 theavailable binding sites are occupied

With O2, we’re dealing with volatile substance,use partial pressure as substitue for

concentration

θ = __[L]__ = __[O2]__ = __pO2__ [L] + Kd [O2] + Kd pO2 + Kd

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Upshot: we can determine the fraction ofbound binding sites by varying [O2] (pO2)

Hyperbolic curve shows that practically all sitesare filled at low pO2 - tight binding of Mb to O2

Fig 7.4b

= ~0.26 kPa

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Protein structure affec ts how ligands bind

Binding of O2 to heme

Fig 7-5b

Steric hindrance inbinding pocketreduces ability ofcompeting substancesto bind

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O2 binding depends on molecular “breathing”

Subtle molecular motions open channels for O2movement

Fig 6.16

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O2 is transported in blood by hemoglobin (Hb)

• Mr = 64,500

• Tetrameric protein; 4 subunits– 2 α chains, 2 β chains, 4 heme groups

– Members of “globin” family– Strong 4°

• Primari ly hydropho bic and H-bond ing

• Key ion pairs (“sal t bridges”) between subunits

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Hb better adapted to O2 transfer, Mb to O2storage

Hb undergoes a structural changeon binding O2

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2 major Hb conformations: “R” and “T” states

R (“relaxed”) - higher affinity for O2

T (“tense”) - lower affinity for O2

Fig 7.10

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Binding of O2 to T state changes positionsof key αα’s surrounding heme

Changes lead to adjustments in ion pairs at αβinterface - narrows pocket between β subunits

Fig 7.11

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Hb binds O2 cooperatively

Must bind O2 efficiently in high [O2] areas(lungs) and release it efficiently in low [O2]

areas (tissues)

High [O 2] (lungs) T R (high af finity)Low [O2] (tissues) R T (low affinity, O2 released

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O2 binding curves for Hb & O2

Hb much more sensitive to O2 concentration

Fig 7.12

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Hb vs. Mb O2 binding curves

• Hb: S-shaped (sigmoidal) curve– Binding of O2 shifts conformation to R state

– Binding of 1s t O2 makes binding of 2nd easier

• Mb: one subunit, one O2 binding site– Each O2 binds independently

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Hb - Classic example of allosteric protein

• Binding of ligand at one site affec ts binding atanother

• Induce conformational changes (activity)

• ligand and “modulator” identical - Homotropicinteraction

• ligand and “modulator” different-Heterotropicinteraction

Some proteins can be both!

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Cooperative ligand binding can be describedquantitatively by the Hill Plot

Fig 7.1326

Hill plots - Cooperative binding of O2 by Hb

Hill coefficient (nH = slope)– nH > 1; positive cooperativity

– nH = 1; ligand binding independent

– nH < 1; negative cooperativity

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Theoretical upper limit of nH = n (no. sites)

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What is the mechanism behind cooperativebinding?

i.e. how does T R

2 models: 1) Concerted

2) Sequential

28Fig 7.14

Concerted Model• Each subunit can exist in 2

conformations (T,R)

• subunits undergotransformationsimultaneously

• No protein has individualsubunits in differentconformations

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Fig 7.14

Sequential model• Ligand binding

inducesconformationalchange in individualsubunits

• Conformationalchanges in onesubunit increaselikelihood of changein 2nd

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Hb also transports H+ and CO2

• Respiration end products to lungs & kidneysfor excre tion

• Bohr effect: H+, CO2 binding inversely relatedto O2 binding

• O2, H+ and CO2 bind at separate sites– O2 - Heme– H+ - αα R groups (His, etc.)– CO2 - N-termini of α and β chains

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Binding of H+ to key His residues form saltbridges - stabilize deoxyhemoglobin T state

High pH environments (low [H+], lungs)

His deprotonated, R state favored - O2 binds

Fig 7.10

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Carbamates formed by CO2 binding alsoform salt bridges which stabilize T state

T state promotes release of O2

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O2 bind ing regu la ted by 2,3-bisphosphoglycerate(BPG)

• Physiological adaptation

to altitude

– @ high altitude, low pO2

• Heterotropic allostericmodification

• Lowers O2 affinity intissues

• Does not signigicantly

alter O2 affinity in lungs

Fig 7.16

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BPG lowers O2 affinity by stabilizing T state

• Significant role in fetal development– Adult Hb: α2β2

– Fetal Hb: α2γ2

– γ subunits lower affinity for BPG

– Enables higher affinity of fetal Hb for O2 -extraction from mother’s blood

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Sickle Cell Anemia - Genetic disease of Hb

• Anemia, blocked capillaries, early death

• Altered formation of Red Blood Cells

• Single αα substitution– Glu Val in 2 β chains

– 2 (-) charges replaced w/hydrophobic “patches”

– Creates “sticky” contact points– Hb molecules form insoluble fibers

– Leads to sickle-shaped cells

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Part II: Complementary Protein-LigandInterac tions: The Immune System

Most protein-ligand interactions don’tinvolve a prosthetic group, such as heme

The binding site is more often a pocket lined with αα Residues arranged to render the

interaction highly specific

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Protein binding sites highly discriminatory -detecting even minor structural differences

among ligands

All vertebrates have an immune system capableof distinguishing “self” from “non-self”

(bacteria, viruses, pathogens, etc.)

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The Immune System - 2 complementary systems

Humoral (fluid) immune system– Soluble proteins (antibodies/immunoglobulins)

• 20% of bloo d protein

– Produced by B-lymphocytes

– Bind bacteria, viruses, large molecules (Mr>5000)and target them for destruction

Cellular Immune system– T lymphocytes, B cells , macrophages

– Recognize infected cells or parasites– TH cells interact w/Macrophages, stimulate B, Tc

& TH cell proliferation

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White blood cells (Leukocytes) of the immunesystem

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Immune system recognition proteins (Ig’sand T cell receptors) specifically bind a

particular chemical structure on their target

Humans can produce over 100,000,000different Immunoglobulins (Ig’s)

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Immune system lexicon

• Antigen– anything capable of eliciting an immune system

response

• Epitope– Molecular structure on antigen to which Ig or T

receptor binds

• Hapten– Small molecule (oligosaccaride, peptide, etc.)

conjugated to large protein which elicits animmune response

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Dinstinguishing “Self” from

“Non-self”

Antigen detection mediated by

Major HistocompatibilityComplex (MHC) proteins

Fig 7.21

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MHC proteins display digested peptide fragmentson the cell surface

• Peptides from proteins digested in cell interior

• Normally from cellular proteins

• During viral infection, viral protein fragments

displayed

• Interac t with T cell receptor to launch immune

response

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2 classes of MHC proteins

MHC I -Found on su rface of nearly all vertebrate ce lls

MHC II - specia lized cells: M acrophages, B ce lls

Fig 7.20

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Tc (cytotoxic) cells undergo selection -preventproduction of cells which recognize “self” MHC’s

Enough T cells survive which carry millions ofdifferent binding specificities

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Interactions at Cell Surface starts Immune Response

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Immune Response

(1) MHC I protein-peptide complexes of infected

cells recognized by specific Tc cells

(2) Tc cells bind & destroy infected cell(perforins)

(3) Tc cells complexed to infected cells producereceptors for interleukins

– Peptides which stimulate cell proliferation48

4) Macrophages & B cells encounter antigen inbloodstream - engulf or bind antigen

5)Macrophages, B cells display antigen on MHC II

6) TH (helper) cells bind to macrophages & B cells– Activated TH cells secrete interleukins to stimulate Tc

& TH production

7) Prolifera ting Tc, TH, B cells & macrophagesdestroy pathogen

8) Remnant of T&B cells mature into memory cells– Basis for long term immunity

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Immunoglobulin (antibody) Binding Sites

• Each Ig has 4 polypeptide chains– 2 “heavy” chains– 2 “light” chains– Linked by noncovalent & disulfide bonds

Fig 7.23

Fig 7.23

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Heavy & Light chains interact to form ‘Y’ structure

• Heavy & Light chains c omposed of “constant” &“variable” regions

– Variable regions create antigen binding site

• Each branch of ‘Y’ has a single antigen binding site

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5 classes of Ig’s: IgA, IgD, IgE, IgG, IgM

• Cell membrane attatched forms

• Secreted forms (milk, tears, saliva)

• Soluble forms, etc. 52

Typical antigen-Ig interactions are very strong!

Kd= [Ig][Ag] ~ 10-10 M! [IgAg]

Fig 7.27

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Biomedical Benefits of Ig-Ag Interaction

Extraordinary binding affinity & specificitymake Ig’s valuable analytical reagents

Fig 7.28

ELISA Assay

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Part III: Protein Interac tions Modulated byChemical Energy: Actin, Myosin & Molecular

Motors

Muscle contraction, Organelle movement,chromosome division - all rely on function of

“motor” proteins fueled by ATP

What stuctural & functional properties allow These proteins to carry out their unique role?

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Motor proteins - exceptional levels of spatial &temporal organization

Myosin– 2 heavy chains, 4 light chains (associate w/head)– C-terminus = “tail”– N-terminus = globular “head” (ATP hydrolysis)

Fig 7.29

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Bundles of myosin form “thick filaments”

• Core of contractile unit (motor)

• “heads” project from both ends

Fig 7.30

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Actin - “thin filaments” (resistance)

• Monomers (G actin) form polymers (F actin)

• “Thin filaments” include troponin & tropomyosin

Fig 7.30

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Each actin monomer can bind to one myosin head

Fig 7.30

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Thick and thin filamentsare organized intoordered structures

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thick & thin filame nts myof ibrils musc le fibers

Each muscle fiber a single, large, mutli-nuclea tedcell - formed from many cells fused together

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Muscle contractile unit = “Sarcomere”

Contains A & I bands– A band = overlapping of thick & thin filaments

– I band = anchor point (“Z” disk) for thin filaments

– Sarcomere extends from 1 Z d isk to the next

Z disk Z diskFig 7.31

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Myosin thick filaments slidealong Actin thin filaments

1) ATP b inds to myosin act in-myosin interaction disrupted acti nreleased

2) ATP h ydrolyzed con formati onchange in myosin myosin bindsto F act in closer to Z disk

3) Pi released my osin conformationchange s trengthen myosin-actininteraction

4) “Power stroke”: myosin pulledtoward Z disk (contract ion)ADP released

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At any given moment, 1-3% of myosin “head”bound to actin - prevents “backsliding”

Interac tion b/w actin/myosin regulated -contraction occurs only in response to

signal from nervous system

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Nerve impulse relases Ca2+ from SarcoplasmicReticulum

• Ca2+ binds troponin

• Causes conformational change in troponin’sinteraction w/ tropomyosin

• Tropomyosin moves, exposes myosin bindingsites on actin

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Binding & Catalysis

• Binding of ligands (ATP,ADP, Pi, Ca2+)causes conformational changes in protein

• Hydrolysis also causes conformationalchanges - results in “power stroke”

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Ch. 7 Summary

• Protein function often entails interac tionw/other molecules (ligands)

• Binding of ligands often induces aconformational change - affecting bindingstrength, catalysis, etc.

• Ligand binding can be regulated by thebinding of other ligands (CO2, BPG, H+, O2)

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• Mb & Hb contain a prosthetic group (heme)which binds O2 reversibly

• Reversible binding can be described byassociation or dissociation constants (Ka, Kd)

• Entry and exit of ligands depends on sterichindrance and molecular motions (“breathing”)

• Adult Hb contain 2 α and 2 β subunits

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• Hb tetramer exists in 2 interchangeable states: T(tense) and R (re laxed)

• T state most stable in deoxygenated state, R moststable in oxygenated state

• Binding of O2 promotes transition from T to Rstate

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• O2 binding to Hb is cooperative & allosteric

– Binding of 1 O2 affects binding of subsequenct O2’s(allostery)

– Binding of 1 O2 enhances binding of subsequent O2’s

(cooperativity)

– Cooperative binding recognized by S-shaped

(sigmoidal) binding curves (θ vs. [L])

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• 2 models proposed to explain cooperativebinding of ligands

(1) Concerted Model

– All subunits in same conformation @ any giventime

– All subunits undergo reversible transition b/w 2states (T vs R) upon binding of ligands

– Succesive binding moves “equilibrium” to highaffinity state

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(2) Sequential Model– Individual subunits undergo conformational

transitions between high and low affinity states uponligand binding

• Hb also binds H+ and CO2 (Bohr effect)– Stabilize T (deoxygenated state)

– O2 binding weakened (release favored)

– Binding of H+, CO2 promotes release of O2 (tissues)– Binding of O2 promotes release of H+, CO2 (lungs)

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• O2 binding also modulated by 2,3 BPG– Stabilizes T state

– Physiological adaptation to altitude

• Immune response mediated by interac tionsamong specialized cells & their associatedproteins (Ig’s, MHC’s, interleukins, receptors)

• MHC proteins display “self” or “non-self”peptides on the cell surface

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• TH cells induce prolifera tion of B & Tc cells

• Humans produce 5 classes of Ig’s– Y-shaped proteins

– Constant & Variable regions

– Variable regions bind to antigens

• Muscle contractions result from organizedinteractions b/w myosin & actin - coupled tothe hydrolysis of ATP

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• Myosin organized into “thick filament” (motor)

• Actin organized into “thin filaments” (resistance)

• ATP hydrolysis coupled to a series ofconformational changes in myosin– Causes thick & thin filament to slide past each other

– Stimulated by release of Ca2+ from nerve impulse