Structure of protein Lecture : 3 - PowerPoint Presentation

Slide1

Structure of protein

Lecture : 3Dr. Shaimaa Munther

Slide2

Learning Outcomes - ProteinsDefine the levels of protein conformation and to indicate the role of weak interactions.Illustrate how protein structure relates to protein function using examples.

Contrast the roles played by peptide bonds with those of amino acid side chains in protein conformation.

Slide3

Structure of ProteinsOVERVIEWThe 20 amino acids commonly found in proteins are joined together

by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a

unique three-dimensional shape.

The

complexity of protein structure is

best analyzed

by considering the molecule in terms of four

organizational levels

,

namely:

Primary

Secondary

Tertiary

Quaternary .

Slide4

Why knowing protein structure is important ????A

protein’s function depends on its specific conformation.In almost every case, the function depends on its ability to recognize and bind to some other molecule.

For example, antibodies bind to particular foreign substances that fit their binding sites.

Enzyme recognize and bind to specific substrates, facilitating a chemical reaction

.

A

functional proteins consists of one or more polypeptides that have been precisely twisted, folded, and coiled into a unique shape.

It is the order of amino acids that determines what the three-dimensional conformation will be.

Slide5

Protein Structure20 Amino Acids

PrimarySecondary

Tertiary

Quaternary

Denatured

Coded

by the DNA

Self assembly to a single (native) structure. Depends on primary structure and solution conditions

Organize

the folding within a single polypeptide.

Arises

when two or more polypeptides join to form a protein

linear sequence

of amino acids

Occurs with

protein degradation

Slide6

Levels of Protein Structure

Slide7

Chemistry of Protein StructurePrimarySecondaryTertiaryQuaternary

Assembly

Folding

Packing

Interaction

Slide8

Note :

Protein Assembly occurs at the ribosome which involves polymerization of amino acids attached to tRNA

, this yields

primary

structure.

Slide9

1- PRIMARY STRUCTURE OF PROTEINS

The sequence of amino acids in a protein is called the primary structure of the protein.

Understanding the primary structure of proteins is

important because

many genetic diseases result in proteins with

abnormal amino

acid sequences, which cause improper folding and loss

or impairment

of normal function.

If

the primary structures of the

normal and

the mutated proteins are known, this information may be used

to diagnose

or study the disease.

Slide10

1- PRIMARY STRUCTURE OF PROTEINS

Linear sequence of amino acid polymer.The precise primary structure of a protein is determined by inherited genetic information.

At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus).

Slide11

PRIMARY STRUCTURE OF PROTEINSIn proteins, amino acids are joined covalently by peptide

bonds.Peptide bond are amide linkages between the α-carboxyl group of one amino acid and the α-amino group of another.

For

example,

valine

and

alanine can

form the dipeptide

valylalanine

through the formation of a

peptide bond.

A. Peptide bond

Slide12

PRIMARY STRUCTURE OF PROTEINS

The peptide bond has a partial double-bond character, that is, it is shorter than a single bond, and is rigid and planar . The peptide bond is generally a trans

bond (instead of

cis

) in large part because of steric interference of the R-groups when in the

cis

position.

Peptide

bonds are

not broken

by conditions

that denature

proteins, such as

heating & high

concentrations of

urea.

Prolonged exposure to

a strong acid or base at elevated temperatures is required to hydrolyze these bonds non enzymically.B.

Characteristics of the peptide bond:

Slide13

The trans-peptide group.

Slide14

1- PRIMARY STRUCTURE OF PROTEINSBy convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N- to the C-terminal end of the peptide. For example, in the previous figure the order of the amino acids is “

valine, alanine.”Linkage of many amino acids through peptide bonds results in an unbranched

chain called a polypeptide

.

Each component

amino acid

in a polypeptide is called a “residue” because it is the portion

of the

amino acid remaining after the atoms of water are lost in the

formation of

the peptide bond

.

When a polypeptide is named,

all amino

acid residues have their suffixes (-ine, -an, -ic

, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal

valine, a glycine, and a C-terminal leucine is called valyl

glycyl leucine.

.

C. Naming the peptide:

Slide15

15

Slide16

1- PRIMARY STRUCTURE OF PROTEINSEven a slight change in primary structure can affect a protein’s conformation and ability to function.

In individuals with sickle cell disease, abnormal hemoglobins, develop because of a single amino acid substitution.These abnormal hemoglobins crystallize, deforming the red blood cells and leading to clogs in tiny blood vessels.

Slide17

2- SECONDARY STRUCTURE OF PROTEINSThe

secondary structure of the polypeptide is a three dimensional structure.It results from hydrogen bond formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen –C=O of another peptide bond.

According to H-bonding there are two main forms of secondary structure:

α-helix

:

It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth

one.

β-sheets

:

It

is

another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment

).

Slide18

Loops & Bends:Turns and bends e.g. : (

-bend) refer to short segments of amino acids that join two units of the secondary structure, such as two adjacent strands of an antiparallel sheet. A turn involves four

aminoacyl

residues, in which the first residue is hydrogen-bonded to the fourth, resulting in a tight 180° turn.

Proline

and

glycine

often are present in turns.

Loops are regions that contain residues beyond the minimum number necessary to connect adjacent regions of secondary structure. Irregular in conformation, loops nevertheless serve key biologic roles. For many enzymes, the loops that bridge domains responsible for binding substrates often contain

aminoacyl

residues that participate in catalysis.

2- SECONDARY

STRUCTURE OF PROTEINS

(



-bend)

Slide19

Secondary structure

α

-helix

β

-sheet

Secondary

structures

α

-helix

and

β

-sheet, have regular hydrogen-bonding patterns.

Slide20

2- SECONDARY STRUCTURE OF PROTEINSα-helix is a spiral structure, consisting of a tightly

packed, coiled polypeptide backbone core, with the side chains of the

component

amino acids extending outward from

the central

axis

to

avoid interfering

sterically

with each other

.

Formed by a H-bond between every 4th peptide bond – C=O to

N-H

Usually

found in

proteins that span a

membrane

The

 helix can either coil to the right or the left.

Each turn of an α-helix contains 3.6 aminoacids.Proline disrupts an

α-helix because its secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix.

Example of

proteins contains α-helices:

keratins are a fibrous proteins. They are a major component

of tissues such

as hair and

skin.

myoglobin, a

globular, flexible

protein molecule

A.

α

-

Helix

Slide21

2- SECONDARY STRUCTURE OF PROTEINSThe β-sheet is another form of secondary structure in which all

of the peptide bond components are involved in hydrogen bonding.

Unlike the α-helix, β-sheets are composed of two or more peptide chains (β-strands), or segments of polypeptide chains, which are almost fully extended.

The

surfaces of β-sheets appear “pleated,” and

these structures

are, therefore, often called “β-pleated sheets.” When

illustrations are

made of protein structure, β-strands are often

visualized as

broad

arrows.

B.

β

-

Sheet

Slide22

Parallel and antiparallel sheets

A β-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains that are arranged either: Anti-parallel – run in an opposite direction of its neighbor (A)

Parallel – run in the same direction with longer looping sections between them (B)

Slide23

α

-helix

β

-sheet

Slide24

Motif: Is a small, specific combinations of secondary structure elements, e.g. -

- loop. The supersecondary

structures (motifs) are produced by packing side chains from adjacent secondary structural elements close to each other.

Motif

Slide25

3-TERTIARY STRUCTURE OF GLOBULAR PROTEINSTertiary structure is determined by a variety of interactions among R groups and between R groups and the polypeptide backbone.

These interactions include: Hydrogen bonds

among polar and /or charged areas

Covalent

(e.g. disulfide) bonding

Ionic bonds

between charged R groups, and hydrophobic interactions

Hydrophobic interactions

among hydrophobic R groups.

Slide26

Examples of bonds/interactions contributing to the tertiary structure of a protein:

two cysteine side chains

Slide27

Slide28

3-TERTIARY STRUCTURE OF GLOBULAR PROTEINSDomain : a compact and self folding component of the protein that usually represents a discreet structural and functional unit.

Polypeptide chains that are greater than 200 amino acids in length generally consist of two or more domains.

The core of a domain is built from combinations of

several motifs usually rich in hydrophobic interactions.

A specialized group of proteins, named

chaperones,

is required for the proper folding of many species of proteins.

Domains

Slide29

Types of Tertiary Structure

Globular

Disordered

Fibrous

Many

soluble

amino acids, protein tends to

minimize surface/volume ratio

Interacts well with water and takes up a random

configuration

Strong secondary

structure allows protein to retain a non-spherical shape

Slide30

Protein folding

Protein folding constitutes the process by which a poly-peptide chain reduces its free energy by taking a secondary, tertiary, and possibly a quaternary structure

Slide31

4- QUATERNARY STRUCTURE OF PROTEINSResults from the aggregation of two or more polypeptide subunits.Collagen is a fibrous protein of three polypeptides that are

supercoiled like a rope.Hemoglobin is a globular protein with two copies of two kinds of polypeptides.

Subunits

may either function independently of each

other, or

may work cooperatively, as in hemoglobin, in which the binding

of oxygen

to one subunit of the tetramer increases the affinity of the

other subunits

for oxygen

.

Subunits are held together by

noncovalent

interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions).

Slide32

QUATERNARY STRUCTURE Any protein consisting of a single polypeptide chain is not in the quaternary structure and, is defined as monomeric

protein. If there are two subunits, the protein is qaternary and is called "dimeric

"

, if three subunits

"

trimeric

"

, and, if several subunits,

"

multimeric

."

Slide33

Quaternary structure of proteins

Hemoglobin

and



chains with

heme

Collagen

triple helix

4 subunits of a

tetrameric

protein

Slide34

The quaternary structure of hemoglobin

Slide35

Protein denaturation results in the unfolding and disorganization of the protein’s secondary and tertiary structures, which are not accompanied by hydrolysis of peptide bonds.

Denaturing agents include heat, organic solvents, mechanical mixing, strong acids or bases, detergents, and ions of heavy metals such as lead and mercury.Denaturation may, under ideal conditions, be reversible, in which

case the protein refolds into its original native structure

when the

denaturing agent is removed. However, most proteins,

once denatured

, remain permanently disordered.

Denatured

proteins

are often

insoluble and, therefore, precipitate from solution.

Denaturation

and

misfolding

of proteins

Slide36

Slide37

Protein denaturation

Slide38

Protein structure: overview

Structural element

Description

Primary

structure amino acid sequence of protein

Secondary

structure helices, sheets, turns/loops

Motfi

association of secondary structures

Tertiary structure folded structure of whole protein

Quaternary structure assembled complex (more than one polypeptide)

2-6

self-contained structural unit

Domain

Slide39

Protein Classification Fibrous

ProteinPolypeptides arranged in long strands or sheetsWater insoluble (lots of hydrophobic AA’s)

Strong but flexible

Structural protein or

contractile proteins

(keratin, collagen,

muscle, microtubules ,

cytoskelton

, mitotic spindle, cilia, flagella

)

Globular

Protein

Polypeptide chains folded into spherical or globular form

Water soluble

Contain several types of secondary structure

Diverse functions

(enzymes,

, haemoglobin,

immunoglobulins, membrane receptor

sites regulatory proteins)

Slide40

Slide41

Analysis of Protein and Amino AcidsLecture :4Dr. Shaimaa Munther

Slide42

1- ANALYSIS OF THE AMINO ACID CONTENT OF BIOLOGIC MATERIALS The first step in determining the primary structure of a polypeptide is to identify and

quantitate its constituent amino acids.PROTEIN

AMINO ACIDS

Slide43

Determination of the Amino Acid Composition of Polypeptide Chain Amino acid analysis involves four basic steps:

hydrolysis of the protein of interest to individual constituent amino acid : this is achieved by treating

protein sample

with hot hydrochloric acid to hydrolyze the peptide bonds. This treatment cleaves the peptide bonds and releases the individual amino acids.

Labeling the amino acids with a detectable UV- absorbing or fluorescent marker e.g.

6-amino-N-hydroxysuccinimidyl

carbamate

, which forms fluorescent derivatives that can be separated by high-pressure liquid chromatography for example.

Slide44

3. Separating the different types of amino acids by chromatographic methods e.g. adsorption , partition or ion-exchange chromatography.4. Measuring and visualization each amino acid type based on the intensity of the detectable marker associated with the emergnce of each type of amino acid from the chromatographic system. Amino acids are visualized usually using

ninhydrin, which forms purple products with amino acids, but a yellow adduct with proline and hydroxyproline. Detection of amino acid is done by amino acid analyzer .

Protrin

sample

Amino Acids

Peptide

Determination of the Amino Acid Composition of Polypeptide Chain

Slide45

Slide46

Slide47

2- Protein Purification and Analysis

Our understanding of protein structure and function has been derived from the study of many individual proteins.To study a protein in detail, the researcher must be able to separate it from other proteins and must have the techniques to determine its properties.A pure preparation is essential before a protein’s properties and activities can be determined.

Methods for separating proteins take advantage of properties that vary from one protein to the next, including size, charge, and binding properties.

Slide48

Steps of Protein PurificationExtraction of protein from tissue Fractionation and purification of protein mixture using multiple purification techniques:

precipitation depending on the differences in relative solubility of individual proteins as a function of pH (isoelectric precipitation), polarity (precipitation with ethanol or acetone), or salt concentration (salting out with ammonium sulfate). Chromatographic techniques separate one protein from another based upon difference in their size (size exclusion chromatography), charge (ion-exchange chromatography), hydrophobicity

(hydrophobic interaction chromatography), or ability to bind a specific

ligand

(affinity chromatography).

Characterization using SDS gel electrophoresis,

isoelectric

focusing or mass spectrometry

Amino acid sequence using

Edman

method or mass spectrometry

Determination of 3D structure using X- ray crystallography.

Slide49

Since proteins contain a number of charged groups, its solubility depends on the concentration of dissolved ionsSalting out is a procedure for seprating and purification of protein mixture by using high concentration of salt.Usually we use ammonium sulphate

salt (NH4)2SO4 since it is a Very Soluble salt that does not harm proteins.

Adding salt at high concentrations lowers the solubility of macromolecules because it competes for the solvent (H

2

O) needed to solvate the macromolecules.

So high [salt] removes the

solvation

sphere from the protein molecules and they come out of solution

Salting Out

: A method of protein separation and purification

Slide50

Salting Out : A method of protein separation and purification

At low ionic strength, all of the proteins are soluble. As the ionic strength increases, the least soluble protein precipitates. At even higher ionic strengths, further proteins precipitate.

This process is continued until the desired protein is precipitated. This process not only allows you to obtain the desired protein, it removes many unwanted proteins in the process

Slide51

Chromatography: A method for protein separation and purification

Chromatography is an analytical methods used to separate molecules. It involves a mobile and a stationary phase. Mobile phase is what the material to be separated is dissolved in.Stationary phase is a porous solid matrix which the mobile phase surrounds.

Separation occurs because of the differing binding/ interactions each molecule has with both the mobile and stationary phase.

Interactions are different depending on the specific chromatographic method used.

Slide52

Types of ChromatographyGas - liquid

: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. Liquid - Liquid: Mobile phase is liquid, stationary phase is liquid usually bound to a solid matrix

.

If separation is based on ionic interaction the method is called:

Ion Exchange Chromatography.

If the separation is base on size of molecule the method is called:

Gel Filtration or Size Exclusion.

If the separation is base on

ligand

affinity the method is called:

Affinity Chromatography.

Slide53

Column ChromatographyThe most powerful methods for fractionating proteins make use of

column chromatography, which takes advantage of differences in protein charge, size, binding affinity, and other properties .A porous solid material with appropriate chemical properties (the stationary phase) is held in a column, and a buffered solution (the mobile phase) percolates through it.

The protein-containing solution, layered on the top of the column, percolates through the solid matrix within the larger mobile phase.

Individual proteins migrate faster or more slowly through the column depending on their properties.

Slide54

Three Chromatographic Methods used in Protein Purification.

Ion-exchange chromatography Size-exclusion chromatography Affinity chromatography

Slide55

1. Ion-Exchange ChromatographyIn ion-exchange chromatography, proteins interact with the stationary phase by charge–charge interactions.

Proteins with a net positive charge at a given pH will tightly adhere to beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge adhere to beads with positively charged functional groups, typically tertiary, or quaternary amines (anion exchangers).

Nonadherent

proteins flow through the matrix and are washed away. Bound proteins are then selectively displaced by gradually raising the ionic strength of the mobile phase, thereby weakening charge–charge interactions.

Proteins elute in inverse order of the strength of their interactions with the stationary phase.

Slide56

Size-exclusion chromatography, also called gel filtration, separates proteins according to size. The column matrix is a cross-linked polymer with pores of selected size.Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and hence take a more direct route through the column. The smaller proteins enter the pores and are slowed by their more labyrinthine path through the column.

2. Size-Exclusion

Chromatography

Slide57

Affinity chromatography separates proteins by their binding specificities.The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads.Enzymes may be purified by affinity chromatography using immobilized substrates, products, coenzymes, or inhibitors.

In theory, only proteins that interact with the immobilized ligand adhere. Bound proteins are then eluted by disrupting protein–ligand

interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentrations.

2. Affinity

Chromatography

Slide58

Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)

The most widely used method for determining the purity of a protein is SDS-PAGE—

polyacrylamide

gel electrophoresis (PAGE) in the presence of the anionic detergent sodium

dodecyl

sulfate (SDS).

Electrophoresis separates charged

biomolecules

based on the rates at which they migrate in an applied electrical field.

For SDS-PAGE,

acrylamide

is polymerized and cross-linked to form a porous matrix.

Slide59

Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)

SDS-PAGE is used to separate protein mixtures in a protein denaturing environment . That is, the SDS causes proteins to denature and take on a rod-like shape and have similar charge to mass ratios.

Since the charge-to-mass ratio of each SDS-polypeptide complex is approximately equal, polypeptides separate based on their relative molecular mass (

Mr

).

Individual polypeptides trapped in the

acrylamide

gel after removal of the electrical field are visualized by staining with dyes such as

Coomassie

blue .

Slide60

Polypeptide SequencingSanger was the first to determine the sequence of a polypeptide.

insulin was the first protein to be sequenced. Sanger won the Nobel prize for protein sequencing.It took 10 years, many people, and it took 100 g of protein!Today it takes one person several days to sequence the same insulin.The most widly used method today for polypeptide sequencing are:

Edman

degradation for short polypeptide chains and mass spectrometry for larger one

Slide61

Edman

Degradation

Sequencing of polypeptide from its N-terminal end

Sequencing is a stepwise process of identifying the specific amino acids at each position in the peptide chain, beginning at the

N­terminal

end.

Phenylisothiocyanate

, known as

Edman's

reagent, is used to label the amino-terminal residue under mildly alkaline conditions

The resulting

phenylthiohydantoin

(PTH) derivative introduces an instability in the N-terminal peptide bond that can be selectively hydrolyzed without cleaving the other peptide bonds.

Edman's

reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle.

This process has been automated ("

sequenator

").

Slide62

Edman Degradation

Slide63

Mass SpectrometryThe superior sensitivity, speed, and versatility of MS have replaced the Edman

technique as the principal method for determining the sequences of peptides and proteins. Mass Spectrometer breaks the peptides down into fragment ions and measures the mass of each piece. MS electrically accelerates the fragmented ions; heavier ions accelerate slower than lighter ones. Thus MS measure mass/charge ratio of an ion. MS can be used to analyze metabolites, carbohydrates, and posttranslational modifications such as phosphorylation or hydroxylation that add identified increments of mass to a protein (Table below).

These modifications are difficult to detect using the

Edman

technique and undetectable in the DNA-derived amino acid sequence.

Slide64

Mass Spectrometry

Slide65