lecture 11 (10/10/17) - boston university · 10/10/17 7 analytical ultracentrifugation –...

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• Reading: Ch4; 125, 132-136 (structure determination)Ch4; 12-130 (Collagen)

• Problems: Ch4 (text); 10, 15

NEXT

• Reading: Ch1; 27-29Ch5; 157-158, 160-161

• Problems: Ch1 (text); 16Ch4 (text); 1, 2, 3, 4, 6, 7, 8

Lecture11(10/10/17)

OUTLINEProtein CharacterizationA. Quaternary structure

1. How determined; a. native sizeb. subunit size

2. UltracentrifugationB. Tertiary structure

1. X-ray diffraction/crystallography2. NMR spectroscopy3. Comparison: NMR versus X-ray crystallography

C. Secondary structure1. Circular dichroism (CD)

D. Collagen1. Special Fibrous Protein: 2. Clues to structure3. 4-S’s4. Biosynthesis 5. Disorders

Lecture11(10/10/17)

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ProteinCharacterization

QuaternaryStructure

MALDI

Determination of Quaternary Structure

Need the “native” molecular weightGel filtrationUltracentrifugation

Need the subunit stoichiometry and molecular weightSDS-PAGEMALDI-MS

Example2:NativeMW=300kDaSubunitMW=75kDa,50kDaSubunitstoichiometry=

(75:50)is2:3

a2b3

Example1:NativeMW=200kDaSubunitMW=50kDa

a4

Example 3: Native MW = 360 kDaSubunit MW = 80 kDa, 40 kDaSubunit stoichiometry = equal

(means with half the size, the smaller one would have to have half intensity on gel)

a3b3

Protein Characterization: Structure Determination

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Protein Characterization: Structure DeterminationExample of correlating band intensity with size an stoichiometry:

If a smaller protein has the same intensity as that of a larger protein, it means the smaller one must be there in larger numbers.

EXAMPLE-5:

~3x

1x

a1b1g1d6

Mr=72, 68, 60, 40 kDa

Native Mr=440 kDa

EXAMPLE-4 IgG:

(but ~½ the size➔6X)

Darker

Lighter

a2b2

Analytical UltracentrifugationIn analytical ultracentrifugation, analytes are separated on the basis of their sedimentation when they experience a centripetal force (Mass,

Shape, Density).Usually carried out at speeds around 60,000 rpm

ThereisaforceduetobuoyancyFb andafrictionalforceonthemoleculeFf,bothwhichopposeitsmovementthroughthemediumFs(centrifugalforceorsedimentaryforce).


~1.4 g/cm3

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Or, described mathematically…Fs + Fb + Ff = Sed. Vel. (rate)

srNf

M s º=-

2

)1(wµrn

coefficient of friction (g/mole•sec)

sedimentation velocity

Specific Volume (occupied by 1 g of solute (cm3/g))

Solvent density in g/mL

Sedimentation coefficient(units of sec)

radius from center of spin

angular velocity

Analytical Ultracentrifugation – Sedimentation Velocity


Analytical Ultracentrifugation – Sedimentation Equilibrium

M is number average molecular weight (mN). If all species are the same, then M = Mr

TWO Types:

srNf

M s º=-

2

)1(wµrn

Sedimentation coefficient(units of sec)

Units of sedimentation are seconds: 1 Svedberg = 1S = 10-13 s

is called the buoyancy factor – and since 1/ approximates , then if the

density of the molecule is close to that of the solution = 0 and the s = 0.

If the molecule is less dense than solution then < 0 and the molecule floats. (lipids or fats in a blood sample)

Proteins are more dense than solution and > 0, and the molecule sediments.

rn-1 n r

rn-1

rn-1



rn-1

𝘥⩟

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The most basic type of ultracentrifugation experiment is to measure the rate at which the molecule moves away from the center of rotation What is actually measured is the movement of the boundary between dissolved molecule and ‘empty’ buffer

t1 t2 t3 t4

Noteshapeofcurves-lesssteepovertimebecauseofdiffusion


Based on mass, shape, and density




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In sedimentation equilibrium, an equilibrium is established between sedimentation away from the center of rotation and diffusion towards the center of rotation (spin at much lower speeds) so we get no boundary between solute and meniscus

Thisisanything thatmeasures

concentration!




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Analytical Ultracentrifugation – Sedimentation EquilibriumDeterminingMolecularWeight

2/)(0,

20

2

)( rrAA eCrC -= s

RTM 2)1( wrns -

=whereCanbedescribedbyequation:

ThisiscanbeexpressedintermsofM:

M =2kT ln

CAC0(r2 – r02)

____________________(1- vr) w2

_



M =2kT ln

CAC0(r2 – r02)

____________________(1- vr) w2

_

Thus,wecangetM.


Butrecall,thisM=mN,anddependsonthenumberaveragemolecularweight.So,iftherearespecieswithdifferentmolecularweights,asinadissociationequilibriumofanoligomericprotein,thisM willbesensitivetothatdissociation.

A2 ⇌ 2A

Kd =[A]2/[A2]

Atmostradii,itsmigrating“heavier”duetoassociation.

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Protein Characterization

Tertiary Structure

Analysisof3o structure*

• X-raycrystallography • NMR

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*and4o structure

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X-rayCrystallographyforProteinStructure


Comparisontoothermethods:

l = 1.54 Å


50


http://www.ibiology.org/ibioeducation/exploring-biology/biochemistry/proteins/3d-structures.htmlX-ray crystallography Primer

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Closestlayersoflinesfromatomsfurthest apartinunitcell

Furtherslayersoflinesfromatomsclosesttogetherinunitcell=resolution

Intensitiesduetointerferenceandamplificationofwaves


Dependsofwhetherwavesareinoroutof

phase

Relationshipofspotsintensityandunitcell[https://www.youtube.com/watch?v=fZ0m8wustVk]


Resolution


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Theelectrondensitymapfromthex-raydiffractiondataisshownasabluecage,andthemodeloftheproteinthat“fits”thisdensityinmodeledinsideofit.

TypicalX-raycrystallography structureProtein Characterization: Structure Determination

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StereoView


NMR for Protein Structure


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NMR for Protein Structure Correlation2Dspectroscopy(COSY)

HHR2

R1 C=O

H H

R2

R1 C=O

Ca

N

Hz ~4-5 Hz ~1-2

J-coupling

F angle


R2

R1

These intensities change depending on f-angle

Can also get f & y angles from secondary chemical shifts (observed from known random coil shifts)(TALOS)

NMR for Protein Structure Correlation2Dspectroscopy(COSY)

HHR2

R1 C=O

H H

R2

R1 C=O

Ca

N

Hz ~4-5 Hz ~1-2

J-coupling

F angle


R2

R1

These intensities change depending on f-angle

Can also get f & y angles from secondary chemical shifts (observed from known random coil shifts)(TALOS)

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NMR for Protein Structure

Nuclear-Overhauser EffectSpectroscopy(NOESY)

61


TypicalNMRstructure

Blue=b-sheet,red=a-helix,green=loopswithout2° structure.

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Blue= N-term,red=C-term“Rainbowcoloring.”


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NMR Structure of a ProteinProtein Characterization: Structure Determination

TypicalNMRstructure

NoticetherearemanyoverlappingstructuresthatallfittheNMRdata.Whereitistight,youhavehigherresolutionandwhereitislooseyouhavepartsofthemoleculethataremoremobile64


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Protein CharacterizationTertiaryStructure

Compare/Contrast X-ray crystallography and NMR:

1) Crystal vs. solution structures the same; not significant crystal constraints

2) NMR not as high resolution3) NMR better at predicting regions that are dynamic; X-ray

uses “B-factors” or even does not show, i.e., “disordered”4) X-ray cannot distinguish “rotomers” of Asn, Gln, Thr; NMR

is unambiguous5) X-ray much better at larger structures; NMR has

assignment problem only good for up to 30-40 kDa 65


Protein Characterization

Secondary Structure

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Ala-Ala Gly-Gly(assuming hard spheres)

(calculation with molecular dynamics including solvent (water))

NAc-Ala-Ala-NH2

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Ala-Ala Gly-Gly(assuming hard spheres)

(calculation with molecular dynamics including solvent (water))

NAc-Ala-Ala-NH2

RamachandranPlot

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SecondaryStructureProtein Characterization: Structure Determination

CD Demo:http://cddemo.szialab.org/

SecondaryStructure


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Collagen

73

3000 Å74

Protein Structure – CollagenExtracellular: cartilage, tendons, bones, teeth, skin, vessels, lungs

Most abundant protein in mammals

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Primary & Secondary structurePrimary structure

(Gly-Pro/Ala-X)330X=hydroxyl-Pro, Glx, Arg, Asx,

hydroxyl-Lys (Cd), Ser

CD of collagen

Hydroxylation reactions catalyzed by proline- and lysine-hydroxylase; require

vitamin C

Amino Acid composition

Amino Acid Sequence

Secondary structureRamachandran plot

f = -65°

y = +130°

Protein Structure – Collagen

random coil


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The 4 S’s for Collagen:

Size ShapeStabilitySolubility


- not

SizeMW = 285,000 DaLong strands (3000 x 14 Å)helix dimensions/parameters

Protein Structure – CollagenStructure F

(°)y(°)

Rise(Dist/residue)

(Å)

Residues/Repeat

Pitch(Distance/repeat)

(Å)

Diameter(Å)

a-helix -57 -47 1.5 3.6 5.4 5.0

Anti- ⇌b-sheet

-139 +135 3.4 2 6.8 -

Parallel ⥤b-sheet -119 +113 3.2 2 6.4 -

b-turn-Type I 4 0 -i + 1 -60 -30 -i + 2 -90 0 -

b-turn-Type II 4 0 -i + 1 -60 120 -i + 2 80 0 -

Collagen -65 +130 3 3 9 14(triple)

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TABLE 4.1 Idealized f and y Angles for Common Secondary Structures in Proteins

Structure f y

a Helix –57˚ –47˚

b Conformation

Antiparallel –139˚ +135˚

Parallel –119˚ +113˚

Collagen triple helix –51˚ +153˚

b Turn type I

i + 1a –60˚ –30˚

i + 2a –90˚ 0˚

b Turn type II

i + 1 –60˚ +120˚

i + 2 +80˚ 0˚


The Collagen Triple Helix

Shape; Collagen-triple helixleft handedright handed twist to three helicesrole of glycinerole of proline


N

C

Allows for packing of triple helix

Locks the helix

The Collagen Triple Helix

Shape; Collagen-triple helixleft handedright handed twist to three helicesrole of glycinerole of proline


N

C

Allows for packing of triple helix

Locks the helix

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Inter-chainH-bonds

G

POH-P

StabilityPacking of GlyInter-stand H-bonds


CollagenmodelpeptidePDBid1CAG

Protein Structure – CollagenPro

Gly

Pro-OH

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MeltingofCollagen

Species BodyTemperature CollagenTmCalf 37 39Shark,barracuda 26 29Cod,deepsearedfish 14 16

StabilityMelting (viscosity or CD vs. temp)cooperativity = sigmoidal plotrole of hydroxyl-proline- Tm

Higher Tm correlates with higher OH-Pro/Pro


50%

Tm=39°

lecture 11 (10/10/17) - boston university · 10/10/17 7 analytical ultracentrifugation –...

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