Protein FunctionProteins do a great deal of the “work” your

body must perform to stay alive. Types of work include specific binding,

catalysis, transport, contraction, & others. In Chapter 5 we look at proteins that

bind/transport O2, that are active in theimmune system, and that cause muscles tocontract.

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Terms/concepts: 1. ligand: something that binds to a protein (or ...) 2. binding site: location on protein where ligand

binds 3. biological binding sites tend to be quite specific 4. proteins are flexible (breathing and much more) 5. induced fit: a way to generate binding energy 6. in multi-subunit proteins, binding at α affects β,

etc. 7. regulation (in this chapter re. binding, but

covalent modifications are also employed) 8. Enzymes: substrate (vs ligand), active (vs site binding)

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I. Reversible binding: Protein-Ligand (O2)

Aside: Three questions1. Why should you care about O2?2. If your blood was devoid of Hb, what would

the [O2] be at saturation (equilibrium?)? (Henry’slaw constant = 1.04 × 10!2 mol'LCatm at 37E C [http://dspace.mit.edu/bitstream/handle/1721.1/32488/61857986.pdf?sequence=1])

3. What is the normal [Hb] for an average healthy20-30 year old human? (See for example: ~14 g/dL inhttp://emedicine.medscape.com/article/2085614-overview Can you convert this to M?)

How much more O2 does your blood carry w/ Hb? We’ll find out that binding isn’t the hard part.

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A. O2 binds tightly to heme (Fe2+)1. Fe ions bind O2, but free Fe ions make highly

reactive species (OH radicals, etc.)2. Porphyrin (tetrapyrrole), see Fig. 5-1

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3. The apo-protein part of heme proteins “directs”binding/catalytic activities of holo-protein: Fig5-2.

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B. Globins: a family of O2-binding proteins1. Have we seen the term “family” before?2. What does that term tell you about sequence

homology (and relatedness) of the globins?3. C. elegans has 33 different globin genes!!!4. In mammals there are usually $ 4 globin

genes.5. Myoglobin is found in what tissues?6. Function:

a) In most mammals: increases in O2 diffusion inmuscles.

b) In cetaceans (What are cetaceans?): an O2 storage

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bank. (What do cetaceans eat?) C. Myoglobin: single binding site for O2.

1. Myoglobin (Mb) has Mr = 16,7002. Single chain, 153 aa residues long3. Globin family is largely α-helical (~78%, all α)4. Nomenclature:

a) Helices named by capitol letters from N terminusb) Turns named by helices they link (eg., CD turn)c) Residues identified by distance from N terminus

or by location within helix (amino terminus: Val1,while the proximal His is His93 a.k.a., His F8)

See Figs. 5-1 to 3.

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Angled view showing all 6 Fe coordination positions

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Myoglobin with α-helical regions shown astubes.

Can you find His93?

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D. Quantitative descriptions of protein-ligandinteraction1. Back to CHM 112.

a) Start by writing balanced chemical reactionb) Write Ka (= Kc) or Kd (1'= Kc) expressionc) Analyze

2. Association of myoglobin and O2:

3. Note that Ka [O2] = MbAO2'Mb

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4. Dissociation of MbAO2:

5. Fractional saturation. θ = [MbAO2][MbAO2] + [Mb]

See Fig. 5-4

6. Can you get from definition of θ to:

[O2] θ = [O2] + Kd

7. Important: When [O2] = Kd, [MbAO2] = [Mb]What would θ be?

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8. Comment on Kd vs. Kp, and pO2

9. What is pO2 in your lungs right now?

E. Protein structure affects how ligands bind1. It’s useful to think of the protein as providing a

specialized environment (diff. from bulk solvent)2. Comment re. different energy pathways in

catalysis3. Mb O2'CO affinity ratio 10 × > than free heme4. Energetic/structure explanation: see Fig. 5-5

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5. Xtal structure interpretation re. O2diffusion options to heme Fe (breathing? You or the protein?)

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F. Hb transports O2 in your body.1. Hb is in erythrocytes = red blood cells (rbcs).2. Mammalian rbcs are enucleate (lifetime . 120 d)

3. [Hb] in rbcs . 34% by weight. (saturated?)4. Arterial blood leaving lungs has θ = ~ 0.965. Venous blood returning to heart has θ = ~ 0.646. So: Roughly 1/3rd of O2 is released during

circulation. (~ 6.5 mL O2 gas released/0.1 L blood)7. Does this bother you?8. Compare contrast Mb, Hb.

a) Structure (including 1E)b) O2 binding properties

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G. Hb subunits (α2β2) are structurally like Mb

1. 1E structure: Sequence (Fig. 5-7) Criticalresidues, α-subunit is missing the D helix

2. 3E structure: See Fig. 5-63. 4E structure: See Fig. 5-8 & pdb 1a3n (Are you

brave enough to look at the “Prot-prot” tab?)

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Deoxy-Hb(human)

pbd code:1a3n

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H. Hb structure changes when O2 binds

1. R (relaxed?) state has higher O2 affinity than2. T (tense/taught?) state 3. At low [O2], T state stabilized (maintained) by

interactions shown in fig. 5-9 (Symmetry?)4. O2 binding induces T!R shift. See Fig. 5-10

a) 1st O2 binding de-puckers heme (Fig. 5-11)b) this causes movement of F helix, & so on...

Important: You need to track the changes onyour own, preferably while looking at 1a3n.

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I. Hemoglobin binds O2 cooperatively

1. Hb problem: bind O2 in lungs (pO2 = 13.3 kPa,in atm?), then release O2 in tissues (pO2 = 4kPa) (See Fig. 5-12)

2. If binding affinity (Ka) is high, you will havevery little release of O2 in tissues

3. If binding affinity is low, Hb will have little O2bound in the lungs to begin with.

4. Comment on the standard binding isotherm,physical limitations imposed on living things.

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5. One way to solve this problem is for Hb tohave multiple personalities. You have alreadymet the two personalities, T and R. Fig. 5-12

6. Hb capacity to shift from T to R on O2 bindingis achieved through subunit interactions.

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J. Quantitative description of cooperativebinding1. Obviously, this requires multiple binding sites2. Chemical reaction:

P + nL W PLn

Does binding occur in individual steps (n1,then n2, etc.) , or is it an “all or nothing”process?

3. On your own, go through the derivation of theHill equation, p. 167.

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log [θ/(1! θ)] = n log [L] ! Kd

y = m x + b

4. See Fig. 5-14, p. 167. For wild type Hb, n . 3. Relate back to the curves in Fig. 5-12.

Remember to look at Box 5-1. Tragic, butinteresting biochemically.

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K. Two models suggest mechanisms forcooperative behavior in binding

1. Concerted model (Monod, Wyman, Changeux)a) All subunits are identicalb) Subunits can exist in more than one statec) The different states are readily interconvertabled) All subunits change states together (concertedly)

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2. Sequential (Koshland) a) Ligand binding induces change in the subunit

doing the binding.b) The bound subunit then induces a change in other

subunits.c) Subunits can change state independently

3. Some people view the concerted model as asubset of the sequential model.

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L. Hemoglobin also transports H+ & CO21. Does transport of CO2 by Hb surprise you?2. Carbonic anhydrase catalyzes the following:

CO2 + H2O W H2CO3 What next for H2CO3?

3. The Bohr (Christian, not Niels) Effect SeeFig. 5-16. What chemical group andinteraction of Hb is critical for this effect to beexhibit? This is a nice example of use of acid-base chemistry to optimize protein function.

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N Hb

H

H

+

O

C

O

H+

N Hb

C

H

O

-O

4. Basis for the Bohr Effect:

HHb+ + O2 W HbO2 + H+ (His146)

5. CO2 binding:a) Carbamate formation allows formation of salt

bridges that stabilize the T (low O2 affinity) state.

Happy with the charge on α-amino?

b) This stimulates release of O2. (Note: H+

production, relate to rxn. in #4 above.25

M. Hb function is influenced by 2,3-bisphosphoglycerate (BPG)

1. Allosteric (other site) binding2. HbBPG + O2 W HbO2 + BPG (mass action?)3. See Fig. 5-17 for quantitative description.4. Comment on BPG and high altitude response,

other hypoxia problems. Fig. 5-18

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N. Sickle-cell anemia: a molecular disease(many are)1. Full explanation/understanding requires more

molecular genetics & population genetics. 2. Protein structure: Glu6 ÿ Val6 in the β-chain3. Protein function: Mutant chains are more

hydrophobic in the region of the substitution.4. What do you know is more likely now? Earlier

question about [Hb] being near saturation?5. Physiological outcome of HbS insolubility?

Figs. 5-19 & -20.

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Note: Structural differences shown in β-subunitsabove are somewhat stylized. They are trying toemphasize surface area contact increase betweensubunits.

6. Distribution, origins of HbS?

7. Why is the gene frequency relatively high?

8. Why Glu ÿ Val?

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II. Complementary Interactions betweenProteins & Ligands: Immune System &Immunoglobulins

Comment on self vs. non-self recognition.

A. Immune response features a specializedarmy of cells and proteins.

1. Terms: humoral, cellular, antigens, epitope,hapten, antibodies (immunoglobulins [IgX’s]),B cell, T cell

2. Nice summary in Table 5-2

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B. Self distinguished from nonself by cellsurface peptide display1. This is mediated by the major histocompatibility complex (MHC) (Coma!)

a) Class I I) Widely distributedii) Display degraded peptide fragments (cell proteins )

b) Class III) Found on macrophages and B lymphocytesii) Display degraded foreign peptide fragmentsiii) Relation to clonal selection

C. Antibodies (ab) have 2 (IgGs) identicalantigen binding sites1. See IgG, Fig. 5-232. IgM, IgD, IgE, IgA

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D. Ab bind tightly specifically to antigen (ag)1. Induced fit (Fig. 5-25)2. Kd’s as low as 10!10

E. Ag-ab interaction and analytical/clinicalchemistry (Fig. 5-26 a), shows general basis1. Clonality (polyclonal, monoclonal)2. ELISA (Fig. 5-26 b)3. Immunoblot (Fig. 5-26 c)

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III. Protein Interactions Modulated byChemical Energy: Actin, Myosin, &Molecular Motors Comment on movement andliving things. (All things that perform meiosis and mitosis!)

A. Major proteins of muscles: myosin & actin

B. Other proteins organize the thin and thickfilaments

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C. Myosin thick filaments slide along actinthin filaments. Functional cycle from Fig 31

1. ATP binding causes release of myosin headfrom actin filament. (Molecular details,analogy?)

2. ATP hydrolysis causes shift of myosin head toa high energy conformation. (Likecompressing or stretching a spring.)

3. Myosin head rebinds actin filament, withconcomitant release of Pi.

4. Pi release initiates return of myosin head to lowenergy state (spring return to low energy state).

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5. ADP is released from myosin head, which getsback to start of the cycle of catalysis, but note myosin filament has moved relative to actinfilament distance of 1 myosin head “swing.”

To summarize, what 3 main things does the myosinhead do?

1. 2. 3.

Please review other parts of Section 5.3 carefullyon your own and see me if you have any questions.

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