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CH339K. Proteins: Primary Structure, Purification, and Sequencing. a -Amino Acid. a. All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules. I prefer this layout, personally…. 2 Amides.

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ch339k

CH339K

Proteins: Primary Structure, Purification, and Sequencing

slide3

All amino acids as incorporated are in the L-form

    • Some amino acids can be changed to D- after incorporation
    • D-amino acids occur in some non-protein molecules
slide17

A Modified Amino Acid That Can Kill You

Histidine

Diphthamide

(2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)

slide18

Diphthamide Continued – Elongation Factor 2

  • Diphthamide is a modified Histidine residue in Eukaryotic Elongation Factor 2
  • EF-2 is required for the translocation step in protein synthesis
slide19

Corynebacteriumdiphtheriae

Corynebacteriophage

diphtheria toxin action
Diphtheria Toxin Action

Virus infects bacterium

Infected bacxterium produces toxin

Toxin binds receptor on cell

Receptor-toxin complex is endocytosed

Endocytic vessel becomes acidic

Receptor releases toxin

Toxin escapes endocytic vessel into cytoplasm

Bad things happen

diphtheria toxin action1
Diphtheria Toxin Action
  • Diphtheria toxin adds a bulky group to diphthamide
  • eEF2 is inactivated
  • Cell quits making protein
  • Cell(s) die
  • Victim dies
slide28

Polymerization

DG0’ = +10-15 kJ/mol

slide29

In vivo, amino acids are activated by coupling to tRNA

Polymerization of activated a.a.:

DGo’ = -15-20 kJ/mol

slide30

In vitro, a starting amino acid can be coupled to a solid matrix

  • Another amino acid with
    • A protected amino group
    • An activating group at the carboxy group
  • Can be coupled
  • This method runs backwards from in vivo synthesis (C N)
cis trans isomerization in prolines
Cis-trans isomerization in prolines
  • Other amino acids have a trans-cis ratio of ~ 1000:1
  • Prolines have cis:trans ratio of ~ 3:1
  • Ring structure of proline minimizes DG0 difference
physical methods or
Physical Methodsor

How to Purify and Sequence a

Weapons-Grade Protein

first question
First Question

How do I measure the amount of protein I have?

beer lambert law
Beer-Lambert Law

c = concentration

l = path length

e = extinction coefficient

An Absorbance = 2 means that only 1% of the incident beam is getting through.

transmittance and absorbance
Transmittance and Absorbance

Absorbance vs. Concentration

Transmittance vs. Concentration

second question
Second Question

How can I spot my protein in the great mass of different proteins?

slide50

The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so:

i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein

agarose
Agarose

Gelidium sp.

sds page
SDS PAGE

Sodium Dodecyl (Lauryl) Sulfate

SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein

carrier ampholytes
Carrier Ampholytes
  • Amphoteric Electrolytes
  • Mixture of molecules containing multiple amino- and carboxyl- groups with closely spaced pIs
  • Partition into a smooth, buffered pH gradient
isoelectric focusing
Isoelectric Focusing

Below the pI, a protein has a positive charge and migrates toward the cathode

Above the pI, a protein has a negative charge and migrates toward the anode

isoelectric focusing foot flesh extracts from pomacea flagellata and pomacea patula catemacensis
Isoelectric FocusingFoot Flesh Extracts from Pomacea flagellata and Pomacea patula catemacensis
slide71

STOP

HERE

protein purification steps
Protein Purification Steps

1 unit = amount of enzyme that catalyzes conversion of 1 mmol of substrate to product in 1 minute

georgi markov
Georgi Markov

1929-1978

ricin
Ricin

Ricin B chain

(the attachment bit)

ricin uptake and release
Ricin uptake and release

endocytosis by coated pits and vesicles or,

endocytosis by smooth pits and vesicles. The vesicles fuse with an endosome.

Many ricin molecules are returned to the cell surface by exocytosis, or

the vesicles may fuse to lysosomes where the ricin would be destroyed.

If the ricin-containing vesicles fuse to the Trans Golgi Network, (TGN), there ís still a chance they may

return to the cell surface.

Toxic action will occur when RTA, aided by RTB, penetrates the TGN membrane and is liberated into the cytosol.

ricin action
Ricin Action
  • Ricin and related enzymes remove an adenine base from the large ribosomal RNA
  • Shut down protein synthesis
slide81
The possibility that ricin might be used as an asymmetric warfare weapon has not escaped the attention of the armed services.

The last time I was qualified to know for sure, there were no effective antidotes.

slide83

Raw

Extract

(NH4)2SO4

Cut

Affinity

Gel

Filtration

salting in salting out
Salting In – Salting out
  • salting in: Increasing ionic strength increases protein solubility
  • salting out: Increasing further leads to a loss of solubility
salting in salting out1
Salting in – salting out

The solubility of haemoglobin in different electrolytes as a function of ionic strength.

Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47

slide86

Salting in: Counterions help prevent formation of interchain salt links

Solubility reaches minimum at pI

slide89

Lyotropic  ChaotropicSeries

Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+>Al3+> guanidinium / urea

Anions: SO42−> HPO42−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3−> ClO4−> SCN−

slide90

Bring to 37% Saturation – ricin still soluble, many other proteins ppt

  • Collect supernatant
  • Bring to 67% Saturation – ricin ppt, many remaining proteins still soluble
  • Collect pellet
  • Redissolve in buffer
slide92

Raw

Extract

(NH4)2SO4

Cut

Affinity

Gel

Filtration

slide93

Separation by chromatography

Basic Idea:

You have a stationary phase

You have a mobile phase

Your material partitions out between the phases.

structure of agarose
Structure of Agarose

Agarose is a polymer of agarobiose, which in turn consists of one unit each of galactose and 3,6-anhydro-a-L-galactose.

Ricin sticks to galactose, so store-bought agarose acts as an affinity column right out of the bottle, with ricin binding the beads while other proteins wash through.

slide98

Raw

Extract

(NH4)2SO4

Cut

Affinity

Gel

Filtration

you knew i couldn t leave it at that
You knew I couldn’t leave it at that…

Vm = matrix volume

Vo = void volume

Vp = pore volume

Vt = total volume

Ve = elution volume

(1a) Vt = Vo + Vp or

(1b) Vp = Vt - Vo

(2) Ve = Vo + Kav*Vp

Combining 1b with 2

slide104

a and b represent the effective separation range

  • c corresponds to the exclusion limit
slide106

Note:smaller = slower, whereas in SDS-PAGE, smaller = faster.

Note

Fig. 3. Measurement of molecular weight of native NAGase enzyme of green crab by gel filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase (filled circle).

From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) Biochemistry (Moscow)71(Supp. 1) 855-859.

slide108

Raw

Extract

(NH4)2SO4

Cut

Affinity

Gel

Filtration

slide110

Bovine Insulin

21 residue A chain

31 residue B chain

Connected by disulfides

In order to sequence the protein, the chains have to be separated

chain separation
Chain Separation
  • Interchain disulfide broken by high concentrations of bME
  • Chains are about the same size – but can take advantage of different pIs
    • B-Chain pI ~ 5.3
    • A-Chain pI ~ 7.2
slide115

Apply bME – treated ricin to DEAE-cellulose at pH 7

  • At pH 7:
    • A chain (pKa 7.2) is essentially uncharged,
    • B chain (pKa 4.8) is highly negative
  • A chain washes through the column
  • B chain sticks, eluted with gradient of NaCl
2 d electrophoresis an aside
2-D Electrophoresis (an aside)
  • Can use two different properties of a protein to separate electrophoretically
  • For analysis of cellular protein content, often use 2-dimensional electrophoresis:
  • 1st dimension is isoelectric focusing
  • 2nd dimension is SDS PAGE
2 d electrophoresis cont
2-D Electrophoresis (cont.)
  • Can use other protein properties to separate
    • Simple PAGE at 2 different pHs
    • PAGE and SDS PAGE
c terminal sequencing
C-Terminal Sequencing
  • Carboxypeptidases are enzymes that chew proteins from the carboxy terminus
  • Can incubate a protein (preferably denatured – more later) with a carboxypeptidase
  • Remove aliquot at intervals (time course)
  • Run amino acid analysis of aliquots
slide127

C-Terminal Sequencing of Rat Plasma Selenoprotein

From Himeno et al (1996) J. Biol. Chem. 271: 15769-15775.

slide130

Sequence differences among vertebrate hemoglobins

Time of Divergence

|-------------|-------------|------------|------------|-------------|------------|

┌───────────────────────────────Shark

│ ┌─────────────────────Perch

└─────────┤

│ ┌─────────────Alligator

└───────┤

│ ┌──────Horse

└──────┤

│ ┌───Chimp

└──┤

└───Human

|-------------|-------------|------------|------------|------------|------------|------------|------------|

Sequence Difference

neutral theory of molecular evolution
Neutral Theory of Molecular Evolution
  • Kimura (1968)
  • Mutations can be:
    • Advantageous
    • Detrimental
    • Neutral (no good or bad phenotypic effect)
  • Advantageous mutations are rapidly fixed, but really rare
  • Diadvantageous mutations are rapidly eliminated
  • Neutral mutations accumulate
what happens to a neutral mutation
What Happens to a Neutral Mutation?
  • Frequency subject to random chance
  • Will carrier of gene reproduce?
  • Many born but few survive
    • Partly selection
    • Mostly dumb luck
  • Gene can have two fates
    • Elimination (frequent
    • Fixation (rare)
genetic drift in action
Genetic Drift in Action

Our green genes are evolutionarily superior!

Never mind…

Ow!

simulation of genetic drift
Simulation of Genetic Drift
  • 100 Mutations x 100 generations:
    • 1 gets fixed
    • 2 still exist
    • 97 eliminated (most almost immediately)
rates cont
Rates (cont.)
  • Slow rates in proteins critical to basic functions
  • E.g. histones ≈ 6 x 10-12 changes/a.a./year
slide139

Rates(cont.)

  • Fibrinopeptides
  • Theoretical max mutation rate
  • Last step in blood clotting pathway
  • Thrombin converts fibrinogen to fibrin
rates cont1
Rates (cont.)
  • Only constraint on sequence is that it has to physically be there
  • Fibrinopeptide limit ≈ 9 x 10-9 changes/a.a./year
relationships among plant hemoglobins arredondo peter raul et al 1998 plant physiol 118 1121 1125
Relationships among plant hemoglobinsArredondo-Peter, Raul, et al (1998) Plant Physiol. 118: 1121-1125
slide144

Phylogenetic trees built from the amino acid sequences of type 1 RIP or A chains (A) and B chains (B) of type 2 RIP (ricin-A, ricin-B, and lectin RCA-A and RCA-B from castor bean; abrin-A, abrina/b-B, and agglutinin APA-A and APA-B from A. precatorius; SNAI-A and SNAI-B, SNAV-A and SNAV-B, SNAI'-A and SNAI'-B, LRPSN1-A and LRPSN1-B, LRPSN2-A and LRPSN2-B, and SNA-IV from S. nigra; sieboldinb-A, sieboldinb-B, SSAI-A, and SSAI-B from S. sieboldiana; momordin and momorcharin from Momordica charantia; MIRJA from Mirabilis jalapa; PMRIPm-A and PMRIPm-B, PMRIPt-A and PMRIPt-B from Polygonatum multiflorum; RIPIriHol.A1, RIPIriHol.A2, and RIPIriHol.A3 from iris hybrid; IRAr-A and IRAr-B, IRAb-A and IRAb-B from iris hybrid; SAPOF from S. officinalis; luffin-A and luffin-B from Luffa cylindrica; and karasurin and trichosanthin from Trichosanthes kirilowii)

Hao Q. et.al. Plant Physiol. 2010:125:866-876

slide145

Phylogenetic tree of Opisthokonts, based on nuclear protein sequencesIñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz Lang (2008) Molecular Biology and Evolution, Jan 9