Lec4_1a Biochemistry 2014 UW

8/9/2019 Lec4_1a Biochemistry 2014 UW

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Monday, October 2nd, 2013

Biochemistry 405 Lecture #4

PROTEIN CHARACTERIZATION

Kane Hall 13010:30- 11:20 am

Lecturer: Wim Hol

Slide Set #2:

Only slide # 33 with suggested Problems updated


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Proteins differ tremendously in size and properties

Several properties are useful for protein purification Specif ic properties are used for protein characterization

Different proteases have different specificit ies

Mass spectrometry is a powerful analytical method

Protein sequences reveal evolutionary relationships

The rate of evolution of different proteins differs

BIG PICTURE ITEMS


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Gly

Ser

ASP

His

Ala

Val

Ile

Leu

Met

Phe

Tyr

Pro

Gln

Cys

Thr

AsnGlu

Lys

Arg

Trp

The Twenty Amino Acids Ultra-schematic

The Building Blocks of All Proteins

neutral

hydrophillic

hydrophobic


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Linking amino acids by forming peptide units.The order of the amino acids is called the Primary Structure of a protein

A Polypeptide ChainDriven by the hydrophobic effect


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How many proteins are possible?

The average protein chain is ~ 400residues in length.

At each position any of the 20 amino acids

could occur, so that the number ofpossibilities is: 20400 = 2.6 x 10520.

The number of atoms in the universe is

estimated as 9 x 1078.

So the number of possible proteins oflength 400 residues would exceed the

universe in size by many orders ofmagnitude.

Protein sizes range from~ 30 to ~ 35,000 amino acids

VVP 3/e Fig on p. 91


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Proteins chains differ considerably in length

From the third column you see that proteins often contain mult iple chains.The last column gives only the molecular mass of a single chain.

You have to know that the average Molecular Mass of an amino acid residue is about 110.

(You do not need to know the names and numbers in the Table unless they come back explicitly in later lectures)


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Protein Production

Very often large amounts of proteins are needed for e.g. 3D

structure determination or drug screening: these usually require

multi-milligram quantities of pure protein.

One can:

1. obtain protein from natural sources

2. clone and overexpress a gene from one species in a rapidly

growing cell of another species: heterologous expression .

Very popular these days are expression systems in-Escherichia coli

-Saccharomyces cerevisiae, Pichia pastoris

- insect cell lines in culture.

- human cell lines in culture


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Protein purif ication procedures

Characteristic Purif ication Procedure

Solubility Salting outOften called: ammonium sulphate fractionation

Size Selective Dialysis Gel filtration chromatography

Charge Ion exchange chromatography

Binding Specificity Affinity chromatography


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VVP 3/e Fig. 5-5

Protein purification based on solubility differences

a: mixture of three proteinsb: salt added, and centrifuged: the red protein is precipitated and in the pellet.c: more salt added, and again centrifuged: the green protein is precipitated and in the pellet.

In this (highly idealized!) manner the mixture can be separated into three pure proteins.In practice, one is already very happy if one protein can become more pure from a mixture.

The most commonly used salt is ammonium sulphate. Hence this method is often called ammonium sulphate fractionation.


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VVP 3/e Fig 2-14

Dialysis can be handy to remove smaller proteins from a mix

Dialysis is normally used for buffer exchange, but the newer membranes are produced with varioussize cut-off limits which allow for removal of proteins below a certain molecular weight..

Entropy driven,

red one can

leave the

dialysis pouch,

blue one cannot


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Gel filtration chromatography

Gel beads used have cavities which are permeable to smaller molecules andimpermeable to larger molecules

Larger molecules come first off the column, smaller molecules are retarded and come later.


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Isoelectric Point, aka as the pI

The isoelectric point of molecule is the pH at which the net charge of the molecule is zero.

The pI of a protein molecule obviously depends primarily on its amino acid composition.However, since the pKs of indiv idual functional groups in a folded protein depend also on

the environment of the group, the pI of a protein depends also on its conformation.Hence, the precise calculation of the pI of a protein is qui te a challenge.

YET : proteins wi th dif ferent overall charge run wi th dif ferent speed in an electrical field, which allows forcharacterization and purification methods based on charge.

If the pH is above the pI

the overall charge of the

protein is negative.

If the pH is below the pI

the overall charge of the

protein is positive.


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Principle of ion exchange chromatography.

Chemical groups R+ on a resin in a column are equil ibratedwith anions A- at low ionic strength. R+A- ion pairs remain onthe column.

Polyanion Pn- (i.e. a protein with an overall charge of -n) is thenadded to the column.

R+A- + Pn- R+Pn- + A-

Pn- is attached to the column matrix and excess A- flushes out.

The column is then washed with several volumes of Na+A- atlow concentration to elute weakly bound impurities.

Now the column is washed with Na+

A-

at higher concentrationwhich elutes the bound Pn- :

R+Pn- + A- R+A- + Pn-

The Pn- polyanion (i.e. the protein) is collected in a fraction

collector.

Anion exchange:


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Matrix is either cellulose or agarose.Popular groups attached to the matrix:

Practice (schematic) of ion exchange chromatography

DEAE = diethylaminoethyl : anion exchange

CM = carboxymethyl : cation exchange


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Affinity chromatographyA very power fu l method but column preparation can be time consuming and/or pricey

1

2

3

4

Once impuri ties are washed off ,

elute desired protein (orange)

with ligand to remove it from

the column.

Impurities coming out with the wash 5

6

Green and purple has no affinity to

the orange compound.

Goal: to elute the yellow


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Protein concentration determination and A280

Proteins usually contain several Trp or Tyr or Phe residues.The side chains of Trp and Tyr absorb quite strongly UV light at 280 nm

Hence, ideal for concentration determination by absorpt ion spectroscopyHence this method is often used.

But not all proteins contain Trp or Tyr or Phe and then other methods are neededAnd the UV method is not very sensitive, rarely lower than 50 to 100 g per mL

(Note that the vertical scaleis logarithmic)


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Protein concentration determination

Coomassie brilliant blue binds to proteins.In acidic solutions, the absorbance shif ts from 465 to 595 nm upon binding to proteins.

Hence the 595 nm absorbance provides a way to measure the total protein concentration.In the so-called Bradford assay, which uses this absorpt ion shi ft of Coomassie,

protein concentrations as low as 1 g per mL can be detected


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Concentration determination of a specific protein

It is often important to find a rapid &reliable assay for the specific protein you

wish to purify.

A popular method is:

Antigenic specificity such as ELISA

(Enzyme-Linked Immunosorbent Assay)

(But you need to generate specific antibodies)

The principle is shown on the right.


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Protein characterization methods

Characterist ic Characterization method

Size Gel filtration chromatography(1)

SDS-PAGE chromatography

Charge Ion exchange chromatography(1)

Binding specificity Enzyme-linked Immunosorbent Assay (ELISA) (1)

Enzyme specificity(2)

Amino acid sequence Mass spectrometry

(1)Already discussed on previous slides in this lecture(2) Will be discussed in this lecture and later lectures


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Amino Acid Sequence

Determination Methods

1. Chemical (explained indetail in book; you dont need

to know those details)

2. By Mass Spectroscopy

(aka as Mass Spec )

In general: based on a divide-and-conquer

approach


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You have to know the protease names and specificities not the source.

Some proteases and their specificity

Remember enzyme for exam


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Mass spectrometry

- Mass determination of purif ied proteins

Electrospray , MALDI and Fast Atom Bombardment techniques.- Requires only picomoles (10-12 mole) of material.

- Time required is very short.

- Mass is accurate to 1 Dalton for proteins to 300 kD.

- Peptide sequencing using tandem mass spectrometry.

- Proteases are employed to fragment the protein into peptides.

- Sequences are determined by matching the masses observed

to expected peptide masses for 1,2,3,... residues


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Electron Spray Ionization Mass Spectrometry (ESI)

Dry N2or some other gas promotes the evaporation of solvent from charged droplets

containing the protein of interest, leaving gas-phase ions, whose charge is due to the

protonation of Arg and Lys residues, thereby yielding so-called (M + nH)n+ ions.

The mass spectrometer then determines the mass-to-charge (m/z) ratio of these ions.

The resulting mass spectrum consists of a series of peaks corresponding to ions that

differ by a single ionic charge and the mass of one proton.

Nifty droplet

making device


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Electron Spray Ionization Mass Spectrometry (ESI)

The ESI-MS spectrum of horse heart apomyoglobin (myoglobin that lacks its heme group).

The measured m/z ratios and the inferred charges for most of the peaks are indicated.

The data provided by this spectrum permit the mass of the original molecule to be calculated.

(See Sample Calculation on VVP 3rd Ed p. 111 or VVP 4th Ed p. 113 if you really interested.

It is great fun. But you dont need to know for the exam).

The peaks all have shoulders because the polypeptide's component elements contain small mixtures

of heavier isotopes (e.g., naturally abundant carbon consists of 98.9%12

C and 1.1%13

C, and naturallyabundant sul fur consists of 0.8% 33S, 4.2% 34S, and 95.0% 35S).


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Sequence information is extremely useful, when placed in the broadcontext of known sequences. For instance for:

- checking if a protein with a new amino acid sequence might have a

similar fold as a protein with known structure. E.g. if 25% or moresequence identity is observed, and not too many gaps occur in thealignment, the proteins adopt the same overall fold.

- creating a family sequence alignment. Invariant amino acids in the family

are likely to be important for function or for a fold characteristic.

- constructing phylogenetic trees . These can give profound insight intoevolutionary relationships between species.

- estimating evolutionary rates of evolution of proteins. Since not all proteinsare subject to the same evolutionary pressure, due to their different functions,their rates of change in the course of time is not the same.

- discovering the distribution of domains having the same fold and yet quitedifferent sequence, often with a characteristic sequence motif, in a widevariety of otherwise often unrelated multi-domain proteins.

Protein Sequence and Protein Evolution


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Family sequence alignment of cytochrome c (ctd.)


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Phylogenetic Tree of Cytochrome c

Each branch point represents an organism ancestral to the species connected above it.

The number beside each branch indicates the number of inferred differences per 100

residues between the cytochromes c of the flanking branch points or species.


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Rates of evolution of four proteins

Different protein families have clearly remarkably different rates of

change in amino acid sequence during evolution(Horizontal axis according to the fossi l record)


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Useful Problems at end of Chapter 5, 3rd Ed VVP:

1, 7, 12, 14, 17,18

Useful Problems at end of Chapter 5, 4th Ed VVP:

1, 3, 8, 10, 19, 23, 25

Useful Problems at end of Chapter 3, 7th Ed VVP:

13, 17 (was: 10,11,13)


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Lec4_1a Biochemistry 2014 UW