CH339K Proteins: Primary Structure, Purification, and Sequencing

 -Amino Acid 

All amino acids as incorporated are in the L-form All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules D-amino acids occur in some non-protein molecules

I prefer this layout, personally…

2 Amides

The Acidic and the Amide Amino Acids Exist as Conjugate Pairs

Ionizable Side Chains

Hydrogen Bond Donors / Acceptors

Disulfide formation

4-HydroxyprolineCollagen 5-HydroxylysineCollagen 6-N-MethyllysineHistones  -Carboxygultamate Clotting factors DesmosineElastin SelenocysteineSeveral enzymes (e.g. glutathione peroxidase) Modified Amino Acids

A Modified Amino Acid That Can Kill You Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio- propyl)-3H-imidazol-4-yl]propanoate) Histidine

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

Corynebacterium diphtheriae Corynebacteriophage

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

Other Amino Acids

Every  -amino acid has at least 2 pKa’s

Polymerization  G 0 ’ = kJ/mol  G 0 ’ = kJ/mol

In vivo, amino acids are activated by coupling to tRNA Polymerization of activated a.a.:  G o ’ = kJ/mol

In vitro, a starting amino acid can be coupled to a solid matrix In vitro, a starting amino acid can be coupled to a solid matrix Another amino acid with Another amino acid with A protected amino group A protected amino group An activating group at the carboxy group An activating group at the carboxy group Can be coupled Can be coupled This method runs backwards from in vivo synthesis (C  N) This method runs backwards from in vivo synthesis (C  N)

Peptide Bond

Resonance stabilization of peptide bond

Cis-trans isomerization in prolines Other amino acids have a trans-cis ratio of ~ 1000:1 Other amino acids have a trans-cis ratio of ~ 1000:1 Prolines have cis:trans ratio of ~ 3:1 Prolines have cis:trans ratio of ~ 3:1 Ring structure of proline minimizes  G 0 difference Ring structure of proline minimizes  G 0 difference

Physical Methods or How to Purify and Sequence a Weapons-Grade Protein

First Question How do I measure the amount of protein I have?

UV Absorption Spectrophotometry

Beer-Lambert Law c = concentration l = path length  = extinction coefficient An Absorbance = 2 means that only 1% of the incident beam is getting through.

Transmittance and Absorbance Absorbance vs. ConcentrationTransmittance vs. Concentration

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

Electrophoresis

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

Polyacrylamide Gels

Polyacrylamide gel electrophoresis of whole cell proteins of three strains of lactic acid bacteria.

Agarose Gelidium sp.

SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein SDS PAGE Sodium Dodecyl (Lauryl) Sulfate

Constant q/M ratio

Disulfide cleavage

Disulfide cleavage and chain separation +  ME

Isoelectric Point

Isoelectric Focusing

pH

Carrier Ampholytes Amphoteric Electrolytes Mixture of molecules containing multiple amino- and carboxyl- groups with closely spaced pIs Partition into a smooth, buffered pH gradient

Separation by pI

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

STOP HERE

Protein Purification Steps 1 unit = amount of enzyme that catalyzes conversion of 1  mol of substrate to product in 1 minute

Purification visualized

Example: Purification of Ricin

Georgi Markov

Ricinus communis – castor oil plant

Ricin Ricin B chain (the attachment bit)

Ricin uptake and release 1.endocytosis by coated pits and vesicles or, 2.endocytosis by smooth pits and vesicles. The vesicles fuse with an endosome. 3.Many ricin molecules are returned to the cell surface by exocytosis, or 4.the vesicles may fuse to lysosomes where the ricin would be destroyed. 5.If the ricin-containing vesicles fuse to the Trans Golgi Network, (TGN), there ís still a chance they may 6.return to the cell surface. 7.Toxic action will occur when RTA, aided by RTB, penetrates the TGN membrane and is liberated into the cytosol.

Ricin Action Ricin and related enzymes remove an adenine base from the large ribosomal RNA Shut down protein synthesis

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.

Significant Terrorist Incidents Involving Chemical and Biological Agents YearOrganizationAgents 1946 DIN ("Revenge" in Hebrew; also Dahm Y'Israel Nokeam, "Avenging Israel's Blood") (Germany) Arsenic Compounds 1970 Weather Underground (United States) Tried to obtain agents from Ft. Detrick by blackmailing a homosexual serviceman R.I.S.E (United States) Typhoid, diphtheria, dysentery, meningitis and several others to be delivered by aerosol Aliens of America (Alphabet Bomber) (United States) Nerve Agents 1980 R.A.F. (Rote Armee Faktion) (Germany) Botulinum toxin 1984Rajneshee Cult (United States)Salmonella enterica serovar typhimurium 1991 Minnesota Patriots Council (United States) Ricin Aum Shinrikyo (Japan) Bacteria and viral agents, toxins, organophosphorus nerve agents Aryan Nation (United States) Yersinia pestis 1995 The Covenant and the Sword (United States) Ricin 1998 Republic of Texas (United States) Bacterial and viral agents 2001Unknown (United States)Bacillus anthracis Fallen Angel (United States)Ricin

Raw Extract ( NH 4 ) 2 SO 4 Cut AffinityGel Filtration

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 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

Solubility reaches minimum at pI Salting in: Counterions help prevent formation of interchain salt links

Salting out: there’s simply less water available to solubilize the protein.

Different proteins have different solubilities in (NH 4 ) 2 SO 4

Lyotropic  ChaotropicSeries Cations: N(CH 3 ) 3 H + > NH 4 + > K+> Na+> Li+> Mg 2+ >Ca 2+ > Al 3+ > guanidinium / urea Anions: SO 4 2− > HPO 4 2− > CH 3 COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO 3 − > ClO 4 − > SCN −

1)Bring to 37% Saturation – ricin still soluble, many other proteins ppt 2)Collect supernatant 3)Bring to 67% Saturation – ricin ppt, many remaining proteins still soluble 4)Collect pellet 5)Redissolve in buffer

Dialysis and Ultrafiltration (How do you get the salt out?)

Raw Extract ( NH 4 ) 2 SO 4 Cut AffinityGel Filtration

Separation by chromatography Basic Idea: You have a stationary phase You have a mobile phase Your material partitions out between the phases.

Affinity Chromatography

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.

Begin adding 0.2 M Lactose

Raw Extract ( NH 4 ) 2 SO 4 Cut AffinityGel Filtration

Castor Beans contain two proteins that bind galactose

Gel Filtration

Gel Filtration (aka Size Exclusion)

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 You knew I couldn’t leave it at that…

a and b represent the effective separation range c corresponds to the exclusion limit

K av

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

RCA Ricin Gel Filtration Separation of Ricin

Raw Extract ( NH 4 ) 2 SO 4 Cut AffinityGel Filtration

Okay, Now Let’s Sequence the A-Chain

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 Interchain disulfide broken by high concentrations of  ME Chains are about the same size – but can take advantage of different pIs –B-ChainpI ~ 5.3 –A-ChainpI ~ 7.2

Ion Exchangers

Apply  ME – 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) Can use two different properties of a protein to separate electrophoretically For analysis of cellular protein content, often use 2-dimensional electrophoresis: 1 st dimension is isoelectric focusing 2 nd dimension is SDS PAGE

2-D Electrophoresis (cont.) Can use other protein properties to separate –Simple PAGE at 2 different pHs –PAGE and SDS PAGE

Sequencing with Phenylisothiocyanate

Applied Biosystems 492 Procise Protein Sequencer

Chain Cleavage: Cyanogen Bromide

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

C-Terminal Sequencing of Rat Plasma Selenoprotein From Himeno et al (1996) J. Biol. Chem. 271:

Tandem Mass Spectrometry can also be used to determine peptide sequences

MOLECULAR EVOLUTION

Time of Divergence | | | | | | | ┌───────────────────────────────Shark │ │ ┌─────────────────────Perch └─────────┤ │ ┌─────────────Alligator └───────┤ │ ┌──────Horse └──────┤ │ ┌───Chimp └──┤ │ └───Human | | | | | | | | | Sequence Difference Sequence differences among vertebrate hemoglobins

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? 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 Ow! Our green genes are evolutionarily superior! Never mind…

Simulation of Genetic Drift 100 Mutations x 100 generations: 1 gets fixed 2 still exist 97 eliminated (most almost immediately)

Rates of Change

Protein Evolution Rates Different proteins have different rates

Rates (cont.) Slow rates in proteins critical to basic functions E.g. histones ≈ 6 x changes/a.a./year

Rates (cont.) Fibrinopeptides Theoretical max mutation rate Last step in blood clotting pathway Thrombin converts fibrinogen to fibrin

Fibrinopeptides keep fibrinogens from sticking together.

Rates (cont.) Only constraint on sequence is that it has to physically be there Fibrinopeptide limit ≈ 9 x changes/a.a./year

Relationships among plant hemoglobins Arredondo-Peter, Raul, et al (1998) Plant Physiol. 118:

Amino acid sequences of several ribosome-inhibiting proteins

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:

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