1 amino acids, peptides and proteins the amino acids in proteins polypeptides and proteins protein...

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Amino Acids, Peptides and ProteinsAmino Acids, Peptides and Proteins

The Amino Acids in Proteins

Polypeptides and Proteins

Protein Function

Protein Size, Composition and Properties

Four Levels of Protein Structure

Protein Primary Structure

Chromatography and Electrophoresis of Proteins

The Amino Acids in Proteins

Polypeptides and Proteins

Protein Function

Protein Size, Composition and Properties

Four Levels of Protein Structure

Protein Primary Structure

Chromatography and Electrophoresis of Proteins

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Amino acidsAmino acids

All proteins are composed of amino acids.

• Twenty common amino acids.

• All are -amino acids except proline.

• A primary amine is attached to the carbon - the carbon just after the acid group.

HH || R-C-COOHR-C-COOH || NHNH22

GeneralStructure

carbon

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Amino acidsAmino acids

Because an acid and base are both present, an amino acid can form a +/- ion.

H H | |

R-C-COOH R-C-COO-

| | NH2 NH3

+

How well it happens is based on pH and the type of amino acid. Called a zwitterionzwitterion.

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-Amino acids-Amino acids

Except for glycine, the carbon is attached to four different groups - chiral center.

Carbohydrates We use the D-D- form.

Amino AcidsWe use the L-L- form.

COO-

|H3N - C - H | R

COO-

|H3N - C - H | R

+

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Classification of amino acidsClassification of amino acids

The -amino acid group is the same in each.

Classified by the type of side chain.

•Group I. non-polar side chains.

•Group II. polar, uncharged side chains

•Group III. acidic side chains

•Group IV. basic side chains

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Group I. Non-polar side chainsGroup I. Non-polar side chains

HH33CC H

\\ |

HCHC-C-COO-

// |

HH33CC +NH3

valine

HH33CC H

\\ |

HC-CHHC-CH22-C-COO-

// |

HH33CC +NH3

leucine

H |

CHCH33--C-COO-

|

+NH3

alanine

HH33CC H

|| |

HH33C-CHC-CH22-CH-CH-C-COO-

|

+NH3

isoleucine

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Group I. Non-polar side chainsGroup I. Non-polar side chains

H |

CHCH33 -S-CH -S-CH22-CH-CH22-C-COO-

|

+NH3methionine

phenylalanine H |

-CH-CH22-C-COO-

|

+NH3

proline

HH22CC CH-COO-

|| |

HH22CC +NH2

HH22CC

NNHH

H |

CHCH22-C-COO-

|

+NH3tryptophan

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Group II. Polar side chainsGroup II. Polar side chains

tyrosine

H |

-CH-CH22-C-COO-

|

+NH3

HO-HO-

H |

HO-CHHO-CH22-C-COO-

|

+NH3

serine

HOHO H || |

CHCH33-CH-CH-C-COO-

|

+NH3threonine

H |

HH-C-COO-

|

+NH3

glycine

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Group II. Polar side chainsGroup II. Polar side chains

O O H | || | |

HH22N-C-CHN-C-CH22-CH-CH22-C-COO-

|

+NH3

glutamine

OO H | || | |

HH22N-C-CHN-C-CH22-C-COO-

|

+NH3

asparagine

H |

HS-CHHS-CH22-C-COO-

|

+NH3cysteine

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Group III. Acidic side chainsGroup III. Acidic side chains

Based on having a pH of 7.

O O H | || | |-O-C-CHO-C-CH22-CH-CH22-C-COO-

|

+NH3

glutamic acid

OO H | || | |--O-C-CHO-C-CH22-C-COO-

|

+NH3

aspartic acid

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Group IV. Basic side chainsGroup IV. Basic side chains

Based on a pH of 7. H

++ |

HH33N-CHN-CH22-CH-CH22-CH-CH22-CH-CH22-C-COO-

|

+NH3

lysine ++NHNH22 H

| || | |

HH22N-C-N-CHN-C-N-CH22-CH-CH22-CH-CH22-C-COO-

|

+NH3

arginine H |

CHCH22-C-COO-

|

+NH3

histidine

NNNNHH HH

HH

HH

++

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Polypeptides and proteinsPolypeptides and proteins

Proteins are polymers made up of amino acids.

Peptide bondPeptide bond - how the amino acids arelinked together to makea protein.

HH ||

HH22NCCOOHNCCOOH

|| RR

++

HH ||

HH22NCCOOHNCCOOH

|| R’R’

H H O O | | ||||

HH22N - C - C -N - C - C -

|| RR

HH ||N - C - COOHN - C - COOH | | | |H H R’ R’ + H2O

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Here is an example sequence of amino acids in a protein.

It also shows the abbreviations commonly used.

alaala argarg asnasn aspasp glnglncyscys gluglu

phephemetmetlyslysleuleuhishis ileileglygly

propro serser thrthr trptrp valvaltyrtyr

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ResidueResidue - term used to refer to the amino acid once incorporated into a polypeptide

PolypeptidePolypeptide - contain 10-100 residues

ProteinProtein - contain more than 100 residues.

Most peptides and proteins isolated from cells contain between 2 - 2000 residues.

An average amino acid has a weight of 110, so protein molecular weights are in the range of 220 - 220,000 (some are much larger).

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PeptidesPeptides

N-terminalresidue

N-terminalresidue

H H O O | | || ||

HH22N - C - C N - C - C

|| RR

HH ||N - C - COOHN - C - COOH | | | |H H R’’ R’’

H H OO | | || ||- NH - C - C -- NH - C - C - || R’R’

C-terminalresidue

C-terminalresidue

peptidelinkages

peptidelinkages

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Protein functionProtein function

EnzymesEnzymes biological catalysts.

Immuno-Immuno- antibodies of immune system. globulinsglobulins

TransportTransport move materials around -hemoglobin for O2.

RegulatoryRegulatory hormones, control metabolism.

StructuralStructural coverings and support -skin, tendons, hair, nails, bone.

MovementMovement muscles, cilia, flagella.

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Protein size, composition and properties

Protein size, composition and properties

One important property is molecular weight. There are two common ways to calculate it.

•Determine the number of amino acid residues, then multiply by 110 -- the average molecular weight of an amino acid.

•Directly measure the mass of a protein and report it in daltons. One dalton = One atomic mass unit.

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Size of some important proteinsSize of some important proteins

Protein MW Residues

Insulin 6,000 51

Cytochrome c 16,000 104

Hemoglobin 65,000 574

Gamma globulin 176,000 1320

Myosin 800,000 6100

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Protein compositionProtein composition

Proteins can be classified based on the number of polypeptides used

MonomericMonomeric - only a single polypeptide chain is present.

OligomericOligomeric - two or more polypeptide chains are present.

The subunit peptide chains are typically held together with noncovalent bonds.

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Protein compositionProtein composition

Proteins are also classified based on their composition.

Simple proteins Simple proteins - only contain amino acid residues.

Conjugated proteins Conjugated proteins - contain other biomolecules - prosthetic groupsprosthetic groups.

These groups impart additional properties to a protein.

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Example - cytochrome C 550Example - cytochrome C 550

Aggregate of proteins andother structures.

Heme structure

Contains Fe2+

Used in metabolism.

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Protein solubilityProtein solubility

Two categories.Two categories.Determined by the types of amino acid side chains involved.

Water soluble Water soluble - globular proteins

Water insoluble Water insoluble - fibrous proteins.

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Four levels of protein structureFour levels of protein structure

Primary structurePrimary structure

The actual sequence of amino acids in a protein.

Secondary structureSecondary structure

The type of regular repeating structure (-helix, -sheet)

Tertiary structureTertiary structure

Interaction of side chains.

Quaternary structureQuaternary structure

Association of two or more polypeptide chains to form a multisubunit molecule.

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Summary ofprotein structure

Summary ofprotein structure

primary secondary

tertiary quaternary

H H O O | | || ||

HH22N - C - C N - C - C

|| RR

HH ||N - C - COOHN - C - COOH | | | |H H R’’ R’’

H H O O | | || ||- NH - C - C -- NH - C - C - || R’R’

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Determination of primary structureDetermination of primary structure

The first step is to isolate the protein in a pure form from its natural source. Typically, only very small amounts can be obtained.

Total amino acid composition can be determined by hydrolysishydrolysis of the protein. (6M HCl at 100oC).

The amount of each amino acid can then be measured chromatography.

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Protein sequencingProtein sequencing

Methods that determine the order of each amino acid in a protein.

Edman degradation.Edman degradation.

• Method of choice for protein sequencing.

• Relies on a sequential degradation by removing one amino acid at a time from the N-terminus.

• Process can be automated and works with peptides with up to 50 residues.

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Edman degradationEdman degradation

CN N

S

O

H

HR

N C S +

phenylisothiocyanate peptide

H+

phenylthiohyantoin remaining peptide

isolate andreact withadditionalreagent.

NH2 CHR'

CO

N COO-

NCHR

CO

NH

CHR'

CO

N COO-H

N CH S

NH2 CHR

CO

NH

CHR'

CO

N COO-

H

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Edman degradationEdman degradation

Problems with the method.Problems with the method.

• Does not provide 100% yield - resulting in contamination.

• Limited to about 50 cycles so proteins must be cut to smaller sizes. Must rely on enzymes and reagents to cleave a protein at known locations.

• Disulfide bonds between cysteine residues can present problems.

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As of 1998, over 30,000 protein sequences were available in a computer database.

Having such information available makes it possible to study and compare sequence information.

Several biochemical conclusions have been made as a result of studying this data.

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Identification of protein families.Identification of protein families.• Proteins with common sequence features

have similar biological function,• This allow for the characterization of newly

discovered proteins.

Example - protein kinasesExample - protein kinasesEnzymes that catalyze the phosphorylation of amino acid residues.All known protein kinases have the same common sequence region (domain) of 240 residues.

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Evolutionary development of proteinsEvolutionary development of proteins• Comparison of protein types for many

organisms.• Possible to establish taxonomic

relationships.

Example - cytochrome cExample - cytochrome cProtein used in aerobic respiration.It has been determined for over 60 organisms.27 residues are the same for all forms.Other variations indicate evolutionary changes.

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Search for dysfunction.Search for dysfunction.• Normally, all residues in a protein are

identical for a species.• Some individuals may produce a protein

with one or more ‘incorrect’ residues.

Example - sickle cell anemia.Example - sickle cell anemia.Two ‘incorrect’ amino acid residues result in malformed hemoglobin.This causes deformation of red blood cells.

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Three dimensional nature of proteins.Three dimensional nature of proteins.• Sequence data can be coupled with

other methods.• X-ray crystallography can produce 3-D

structural information. It is a difficult method and has not kept up with the number of proteins that have been isolated.

• Sequencing may offer an alternative approach.

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Example - ribonuclease

LysNH2

Glu

Thr

Ala Ser

Ser

Asp

Try

Asp

Glu

Ser

Cys

Met

Met

Lys

Ser

Ala Ala Lys Phe Glu Arg Glu His Met Asp Ser Ser Thr Ser Ala Ala

ArgAspLeuLysAspArgCysLysProValAspThrPheValHis Thr

Glu

Ser

Leu

Ala

Asp

Val

GluAla Val Cys Ser Glu

Lys

Asp

Val

AlaCys

LysAsp

Gly

Glu

Thr Asp

Cys Tyr Glu Ser Tyr Ser Thr Met SerIle

Thr

Asp

Cys

Arg

Glu

Thr

Gly

Ser

Ala Glu Thr Thr Lys Tyr Ala Cys Asp Pro Try Lys Ser

ValAla Cys

Lys

Asp

Ile

Ile

Glu Gly Asp Pro Tyr Val Pro Val His Phe

Ala

Ser

HOOC - Val

Asp

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Example - ribonuclease

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Chromatography and electrophoresis of proteins

Chromatography and electrophoresis of proteins

For a protein to be assayed by X-ray crystallography or protein sequencing, a pure sample must be produced.

After preparation of a cell extract, an appropriate separation method must be employed. Two such methods are:

ChromatographyChromatography

ElectrophoresisElectrophoresis

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ChromatographyChromatography

Several chromatographic methods have been attempted to isolate pure protein fractions.

ion exchangeion exchange

thin layer chromatographythin layer chromatography

column liquid chromatographycolumn liquid chromatography

size exclusion chromatographysize exclusion chromatography

affinity chromatographyaffinity chromatography

Affinity chromatography is becoming increasingly more important.

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Affinity ChromatographyAffinity Chromatography

The method dates back to 1910.

Modern method was first published in 1967, by Axen, et al. -- ‘Cyanogen bromide Method for the Immobilization of Ligands on Agarose.’

Ohlson (1978) was the first to demonstrate the use of a rigid, microparticulate support - beginnings of instrumental method.

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Affinity ChromatographyAffinity Chromatography

The method involves the interaction of a ligand with the solute of interest. It can be viewed as being comparable to ion-exchange.

Two general types of ligandsTwo general types of ligands

SpecificSpecific Binds only to one species.Antibody/antigen

GeneralGeneral Group specificBinds to specific groupson target species.

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Affinity chromatographyAffinity chromatography

SupportSupportThe material that the ligand is bound to.

Ideally, it should be rigid, stable and have a high surface area.

Agarose is the most popular although cellulose, dextran and polyacrylamide have been evaluated.

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Agarose gelAgarose gel

A polymer of D-galactose and 3,6-anhydro-L-galactose.

It can be used at pressures up to 1 psi and over a pH range of 4-9.

Cross-linking can be used to extend the pressure range.

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The separation is conducted in four basic steps.

Sample introductionSample introduction

Adsorption of components of interestAdsorption of components of interest

Removal of impuritiesRemoval of impurities

Elution of components.Elution of components.

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Sample introductionSample introductionYou must make sure that your column has adequate capacity.

ligandspacer matrix

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AbsorptionAbsorptionUsing a slow flow, your sample is then allowed to pass through the column.

The flow helps drive your sample components towards ‘fresh’ sites.

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WashingWashing

Next, you can remove impurities by passing several volumes of fresh solvent through the column.

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ElutionElutionThe component of interest must then be removed and collected.

This also acts to regenerate the column.

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Elution methodsElution methods

BiospecificBiospecificAn inhibitor is added to the mobile phase (free ligand).

The free ligand will compete for the solute.

This approach is most often used when a low molecular weight inhibitor is available.

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Elution methodsElution methods

NonspecificNonspecificA reagent is added that denatures the solute (pH, KSCN, urea, ionic strength...)

Once denatured, the solute is released from the ligand.

If the solute is to be further used, it must not be irreversibly altered.

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Column: 50 mm x 30 mmcontaining 60 ml of Protein A Sepharose

Sample: 5 liter cell culturesupernatant with mouse IgGa2 and 0.5% fetal calf serum.

Starting buffer:0.1 M Na2HPO4, pH 7

Elution buffer:0.1 M citric acid, pH 4

Flow rate: 66.6 ml/min

60 70 80 minutes

An example.

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A separation method that relies on both the size and the charge of a species.

• Samples are placed in an electrical field.

• They tend to migrate to specific positions in the field.

• With gel electrophoresis, a cross-linked polymer acts like a molecular sieve - smaller proteins move faster than larger ones.

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Bands can then be compared to standards as a means of identifying the molecular weight. Band patterns can be used to indicate a protein’s origin.

MW

200,000

100,000

50,000

25,000

12,500

6,250

standard sample

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