11/09/2010enz regulation ii; molecular motors i regulation ii; molecular motors i andy howard...

11/09/2010Enz Regulation II; Molecular Motors I

Regulation II;Molecular Motors

IAndy Howard

Introductory Biochemistry9 November 2010

11/09/2010 Enz Regulation II; Molecular Motors I

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Hemoglobin exemplifies allostery

Even though it isn’t really an enzyme, hemoglobin can teach us how allostery in enzymes works.

After that we’ll talk about molecular motors.


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Globins & Motor Topics Molecular Motors:

Definition Microtubules and their partners Tubulin Structure Cilia & flagella Movement of organelles

Dyneins & kinesins

DNA helicases Bacterial flagella

Globins as Examples Oxygen binding Tertiary structure Quarternary structure

R and T states Allostery Bohr effect BPG as an effector Sickle-cell anemia


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Setting the stage for this story

Myoglobin is a 16kDa monomeric O2-storage protein found in peripheral tissues

Has Fe-containing prosthetic group called heme; iron must be in Fe2+ state to bind O2

It yields up dioxygen to various oxygen-requiring processes, particularly oxidative phosphorylation in mitochondria in rapidly metabolizing tissues


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Setting the stage II Hemoglobin (in vertebrates, at least) is a tetrameric, 64 kDa transport protein that carries oxygen from the lungs to peripheral tissues

It also transports acidic CO2 the opposite direction

Its allosteric properties are what we’ll discuss


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Structure determinations Myoglobin & hemoglobin were the

first 2 proteins to have their 3-D structures determined experimentally Myoglobin: Kendrew, 1958 Hemoglobin: Perutz, 1958 Most of the experimental tools that crystallographers rely on were developed for these structure determinations

Nobel prizes for both, 1965 (small T!)

QuickTime™ and a decompressor

are needed to see this picture.



Photo courtesyOregon State Library

Photo courtesyEMBL


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Myoglobin structure

Almost entirely -helical 8 helices, 7-26 residues each Bends between helices generally short Heme (ferroprotoporphyrin IX) tightly but noncovalently bound in cleft between helices E&F

Hexacoordinate iron is coordinated by 4 N atoms in protoporphyrin system and by a histidine side-chain N (his F8): fig.15.25

Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site

QuickTime™ and aTIFF (Uncompressed) decompressor


Sperm whale myoglobin; 1.4

Å18 kDa monomer

PDB 2JHO


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O2 binding alters myoglobin structure a little

Deoxymyoglobin: Fe2+ is 0.55Å out of the heme plane, toward his F8, away from O2 binding site

Oxymyoglobin: moves toward heme plane—now only 0.26Å away (fig.15.26)

This difference doesn’t matter much here, but it’ll matter a lot more in hemoglobin!


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Hemoglobin structure

Four subunits, each closely resembling myoglobin in structure (less closely in sequence);H helix is shorter than in Mb

2 alpha chains,2 beta chains



Human deoxyHbPDB 2HHB1.74Å65kDa hetero-tetramer


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Subunit interfaces in Hb

Subunit interfaces are where many of the allosteric interactions occur Strong interactions:

1 with 1 and 2,1 with 1 and 2

Weaker interactions:1 with 2, 1 with 2



Image courtesyPittsburghSupercomputingCenter


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Subunit dynamics 1-1 and 2-2 interfaces are solid and don’t change much upon O2 binding

1-2 and 2-1 change much more:the subunits slide past one another by 15º Maximum movement of any one atom ~ 6Å Residues involved in sliding contacts are in helices C, G, H, and the G-H corner

This can be connected to the oxygen binding and the movement of the iron atom toward the heme plane


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Conformational states

We can describe this shift as a transition from one conformational state to another

The stablest form for deoxyHb is described as a “tense” or T state Heme environment of beta chains is almost inaccessible because of steric hindrance

That makes O2 binding difficult to achieve The stablest form for oxyHB is described as a “relaxed” or R state

Accessibility of beta chains substantially enhanced


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Hemoglobin allostery

Known since early1900’s thathemoglobin displayedsigmoidal oxygen-binding kinetics

Understood now to be a function of higher affinity in 2nd, 3rd, 4th chains for oxygen than was found in first chain

This is classic homotropic allostery even though this isn’t really an enzyme


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R T states and hemoglobin

We visualize each Hb monomer as existing in either T (tight) or R (relaxed) states; T binds oxygen reluctantly, R binds it enthusiastically

DeoxyHb is stablest in T state Binding of first Hb stabilizes R state in the other subunits, so their affinity is higher


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Binding and pO2

Hill found that that binding could be modeled by a polynomial fit to pO2

Kinetics worked out in 1910’s: didn’t require protein purification, just careful in vitro measurements of blood extracts



Sir Archibald V. Hill photo courtesy nobelprize.org


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Hill coefficients Actual equation is on next page Relevant parameters to determine are P50, the oxygen partial pressure at which half the O2-binding sites are filled, and n, a unitless value characterizing the cooperativity

n is called the Hill coefficient.


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pO2 and fraction oxygenated

If Y is fraction of globin that is oxygenated and pO2 is the partial pressure of oxygen,then Y/(1-Y) = (pO2 /P50)n

4th-edition formulation: P50n K so

Y/(1-Y) = pO2n / K

P50 is a parameter corresponding to half-occupied hemoglobin work out the algebra: When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2.

Note that the equation on p.496 of the enhanced 3rd edition is wrong!


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Real Hill parameters (p.496)

Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr Perfect cooperativity, tetrameric protein: n =4

No cooperativity at all would be n = 1. Lung pO2 ~ 100 Torr;peripheral tissue 10-40 Torr

So lung has Y~0.98, periphery has Y~0.06! That’s a big enough difference to be functional

If n=1, Ylung=0.79, Ytissue=0.28; not nearly as good a delivery vehicle!


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MWC theory Monod, Wyman, Changeux developed mathematical model describing TR transitions and applied it to Hb

Accounts reasonably well for sigmoidal kinetics and Hill coefficient values

Key assumption:ligand binds only to R state,so when it binds, it depletes R in the TR equilibrium,so that tends to make more R



Jacques MonodPhoto Courtesy Nobelprize.org


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Koshland’s contribution

Conformational changes between the two states are also clearly relevant to the discussion

His papers from the 1970’s articulating the algebra of hemoglobin-binding kinetics are amazingly intricate



Dan KoshlandPhoto Courtesy U. of California


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Added complication I: pH Oxygen affinity is pH dependent

That’s typical of proteins, especially those in which histidine is involved in the activity (remember it readily undergoes protonation and deprotonation near neutral pH)

Bohr effect (also discovered in early 1900’s): lower affinity at low pH (fig. 15.33)



Christian Bohrphoto courtesyWikipedia


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How the Bohr effect happens

R form has an effective pKa that is lower than T

One reason: In the T state, his146 is close to asp 94. That allows the histidine pKa to be higher

In R state, his146 is farther from asp 94 so its pKa is lower.

Cartoon courtesy Jon Robertus, UT Austin


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Physiological result of Bohr effect Actively metabolizing tissues tend to produce lower pH

That promotes O2 release where it’s needed


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CO2 also promotes dissociation High [CO2] lowers pH because it dissolves with the help of the enzyme carbonic anhydrase and dissociates:H2O + CO2 H2CO3 H+ + HCO3

-

Bicarbonate transported back to lungs When Hb gets re-oxygenated, bicarbonate gets converted back to gaseous CO2 and exhaled


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Role of carbamate

Free amine groups in Hb react reversibly with CO2 to form R—NH—COO- + H+

The negative charge on the amino terminus allows it to salt-bridge to Arg 141

This stabilizes the T (deoxy) state


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Another allosteric effector 2,3-bisphosphoglycerate is a heterotropic allosteric effector of oxygen binding

Fairly prevalent in erythrocytes (4.5 mM); roughly equal to [Hb]

Hb tetramer has one BPG binding site BPG effectively crosslinks the 2 chains

It only fits in T (deoxy) form!

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

BPG (Wikimedia)


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BPG and physiology

pO2 is too high (40 Torr) for efficient release of O2 in many cells in absence of BPG

With BPG around, T-state is stabilized enough to facilitate O2 release

Big animals (e.g. sheep) have lower O2 affinity but their Hb is less influenced by BPG


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Fetal hemoglobin

Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG

Fetal Hb is 22; doesn’t bind BPG as much as .

That helps ensure that plenty of O2

gets from mother to fetus across the placenta


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Sickle-cell anemia Genetic disorder: Hb residue 6 mutated

from glu to val. This variant is called HbS.

Results in intermolecular interaction between neighboring Hb tetramers that can cause chainlike polymerization

Polymerized hemoglobin will partially fall out of solution and tug on the erythrocyte structure, resulting in misshapen (sickle-shaped) cells

Oxygen affinity is lower because of insolubility


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Sickling and polymerization


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Why has this mutation survived?

Homozygotes don’t generallysurvive to produce progeny;but heterozygotes do

Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled

BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem



Deoxy HbS2.05 Å

PDB 2HBS


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How is sickling related to malaria? Malaria parasite (Plasmondium spp.) infects erythrocytes

They’re unable to infect sickled cells

So a partially affected cell might survive the infection better than a non-sickled cell

Still some argument about all of this

Note that most tropical environments have plenty of oxygen around (not a lot of malaria at 2000 meters elevation)



Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389


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Other hemoglobin mutants Because it’s easy to get human blood, dozens of hemoglobin mutants have been characterized

Many are asymptomatic Some have moderate to severe effects on oxygen carrying capacity or erythrocyte physiology


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What is a molecular motor?

A protein-based system that interconverts chemical energy and mechanical work

We’ll discuss several molecular motors today, and then next Monday we’ll look (perhaps) at the most important one: the vertebrate muscle.


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Microtubules

30-nm structures composed of repeating units of a heterodimeric protein, tubulin -tubulin: 55 kDa -tubulin: 55 kDa also

Structure of microtubule itself: polymer in which the heterodimers wrap around in a staggered way to produce a tube


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Tubulin structure

and are similar but not identical Structure determined by electron diffraction, not X-ray diffraction

Some NMR structures available too Two GTP binding sites per monomer Heterodimer is stable if Ca2+ present


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iClicker quiz question 1 Why might you expect crystallization of tubulin to be difficult? (a) It is too big to crystallize (b) It is too small to crystallize (c) Proteins that naturally form complex but non-crystalline 3-D structures are resistant to crystallization

(d) It is membrane-bound (e) none of the above


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Tubulin dimer

G&G Fig. 16.2


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Microtubule structure

Polar structure composed of / dimers

Dimers wrap around tube as they move

Asymmetric: growth at plus end


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Treadmilling Dimers added at plus end while others removed at minus end (GTP-dependent): that effectively moves the microtubule

Fig. 16.3 / 16.13


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Role in cytoskeleton Microtubules have a role apart from their role in molecular motor operations:

They are responsible for much of the rigidity of the cytoskeleton

Cytoskeleton contains: Microtubules (made from tubulin) Intermediate fibers (7-12nm; made from keratins and other proteins)

Microfilaments (8nm diameter: made from actin)


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Cytoskeletal components

Fig. 16.4 /16.11


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Cilia and flagella Both are microtubule-based structures used in movement

Cilia: short, hairlike projections, found on many animal and lower-plant cells

beating motion moves cells or helps move extracellular fluid over surface

Flagella Longer, found singly or a few at a time

Propel cells through fluids


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Axonemes

Bundle of microtubule fibers: Two central microtubules Nine pairs of joined microtubules Often described as a 9+2 arrangement

Surrounded by plasma membrane that connects to the cell’s PM

If we remove the PM and add a lot of salt, the axoneme will release a protein called dynein


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Axoneme structure Inner pair connected by bridge

Outer nine pairs connected to each other and to inner pair

Fig. 16.5


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How cilia move

Each outer pair contains asmaller, static A tubule anda larger, dynamic B tubule

Dynein walks along B tubulewhile remaining attached toA tubule of a different pair

Crosslinks mean the axoneme bends Dynein is a complex protein assembly:

ATPase activity in 2-3 dynein heavy chains

Smaller proteins attach at A-tubule end


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Dynein movement Fig. 16.6


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Inhibitors of microtubule polymerization

Vinblastine & vincristine are inhibitors: show antitumor activity by shutting down cell division

Colchicine inhibits microtubule polymerization: relieves gout, probably by slowing movement of white cells


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Paclitaxel: a stimulator

Formerly called taxol Stimulates microtubule polymerization

Antitumor activity Stimulates search for other microtubule polymerization stimulants


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iClicker question 22. How do you imagine paclitaxel might work?

(a) by producing frantic cell division (b) by interfering with microtubule disassembly, preventing cell division

(c ) by causing changes in tertiary structures of and tubulin

(d) none of the above


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Movement of organelles and vacuoles

Can be fast:2-5 µm s-1

Hard to study 1985: Kinesin isolated

1987: Cytosolic dynein found


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Cytosolic dynein Mostly moves organelles & vesicles from (+) to (-), so it moves things toward the center of the cell

Heavy chain ~ 400kDa, plus smaller peptides (53-74 kDa)

Microtubule-activated ATPase activity


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Kinesin Mostly moves organelles from (-) to (+) That has the effect of moving things outward

360 kDa: 110 kDa heavy chains, also 65-70 kDa subunits (2 + 2?)

Head domain of heavy chain (38 kDa) binds ATP and microtubule: cooperative interactions between pairs of head domains in kinesin, causing conformational changes in a single tubulin subunit

8 nm movements along long axis of microtubule

11/09/2010enz regulation ii; molecular motors i regulation ii; molecular motors i andy howard...

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