protein (structures and functions)

8/12/2019 Protein (Structures and Functions)

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Introduction:

Protein

Dyah Kinasih Wuragil

Veterinary Medicine School, Brawijaya University

[email protected]/[email protected]


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DNA

(Genotype)

Protein

Introduction:


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Why Studying Proteins?

They perform many vital functions, e.g.: catalysis of reactions

storage of energy

transmission of signals building blocks of muscles

They are linked to key biological problems that

raise major computational challengesmostly due to their large sizes (100s to several1000s of atoms), many degrees of kinematicfreedom, and their huge number (millions)


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4

PROTEINS

Proteins are polymers made ofmonomers called amino acids

All proteins are made of 20 differentamino acids linked in different orders

Proteins are used to build cells, actas hormones & enzymes, and domuch of the work in a cell


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FOUR TYPES OF PROTEINS

Structural

Contractile

Storage

Transport


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TYPES OF PROTEINS

Type Examples

Structural tendons, cartilage, hair,

nails Contractile muscles

Transport hemoglobin

Storage milk Hormonal insulin, growth

hormone

Enzyme catalyzes reactions in cells

Protection immune res onse


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Proteins are chains of amino acids Polymera molecule composed of repeating

units


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Amino acid composition

Basic Amino AcidStructure:

The side chain, R,varies for each ofthe 20 amino acids

C

R

C

HN

O

OHH

H

Aminogroup

Carboxylgroup

Side chain


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TYPES OF AMINO ACIDS

Nonpolar R = H, CH3, alkyl groups, aromaticO

Polar ll

R =CH2OH,CH2SH,CH2CNH2,

(polar groups withO-, -SH, -N-)

Polar/Acidic

R = CH2COOH, or -COOH

Polar/ Basic

R =CH2CH2NH2


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ESSENTIAL AMINO ACIDS

10 amino acids not synthesized by thebody

arg, his, ile, leu, lys, met, phe, thr, trp,val

Must obtain from the diet

All in dairy products

1 or more missing in grains

and vegetables


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Protein Structure andFunction 11

The Peptide Bond

Dehydration synthesis

Repeating backbone: NCCNCC

Conventionstart at amino terminusandproceed to carboxy terminus

O O


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Protein tructure andFunction 12

Peptidyl polymers

A few amino acids in a chain are called apolypeptide. A proteinis usually composed of 50to 400+ amino acids.

Since part of the amino acid is lost during

dehydration synthesis, we call the units of aprotein amino acid residues.

carbonylcarbon amide

nitrogen


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Side chain properties

Recall that the electronegativity of carbonis at about the middle of the scale for lightelements Carbon does not make hydrogen bonds with

water easilyhydrophobic O and N are generally more likely than C to h-

bond to waterhydrophilic

three general groups of amino acid: Hydrophobic

Charged (positive/basic & negative/acidic)

Polar


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Primary & Secondary Structure

Primary structure = the linear sequenceofamino acids comprising a protein:AGVGTVPMTAYGNDIQYYGQVT

Secondary structure Regular patterns of hydrogen bonding in

proteins result in two patterns that emerge innearly every protein structure known: the -helixand the-sheet

The location of direction of these periodic,repeating structures is known as thesecondary structureof the protein


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Levels of ProteinStructure

Secondary structureelements combine toform tertiary

structure

Quaternary structureoccurs in

multienzymecomplexes

Many proteins areactive only as

homodimers,homotetramers, etc.


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PRIMARY STRUCTURE OF APROTEIN

It is the sequence of amino acids that makeseach protein different from the next

Dipeptide = 2 amino acids

Tripeptide = 3 amino acids

Polypeptide = many amino acids

Most proteins have many 100 amino acids

Peptide Bonds


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Primary structure

This is simply the amino acid sequences ofpolypeptide chains


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NH2 COOH1 NH2 COOH2

NH2 C N COOH

O

H

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Amino acids are connected head to tail

Dehydration-H2O


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SECONDARY STRUCTURE

Local organization of protein backbone: -helix, -strand (which assemble into -sheet),turn and interconnecting loop.

Alignment of polypeptides as a right-hand

alpha helix

Stabilized by hydrogen bonds betweencarboxyl (C=O) and imido (NH) groups


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TERTIARY STRUCTURE

Three dimensional folding and coiling ofpolypeptide into globular 3-D structure

Caused by additional chemical interactionsamong side chains

Disulfide bonds


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QUATERNARYSTRUCTURE

Assembly of homo orheteromeric proteinchains.

Usually the functional

unit of a protein,especially for enzymes

Interactive folding of several polypeptide chainstogether to form a singlefunctional protein

Functional proteins also might incorporate mineralsor other nonprotein components


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Protein Structures or CONFORMATIONS

Hydrogen bondPleated sheet

Amino acid

(a) Primary structure

Hydrogen bond

Alpha helix

(b) Secondarystructure

Polypeptide(single subunit)

(c) Tertiary structure

(d) Quaternary structure


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A proteins function depends on itsspecific conformation

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

It is the order of amino acids that determineswhat the three-dimensional conformation willbe.


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A proteins specific conformation

determines its function. In almost every case, the function

depends on its ability to recognize andbind to some other molecule. For example, antibodies bind to particular

foreign substances that fit their binding sites.

Enzyme recognize and bind to specificsubstrates, facilitating a chemical reaction.

Neurotransmitters pass signals from one cellto another by binding to receptor sites onproteins in the membrane of the receivingcell.


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A proteins conformation can change in responseto the physical and chemical conditions.

Changes in pH, salt concentration, temperature,or other factors can unravel or denatureaprotein. These forces disrupt the hydrogen bonds, ionic

bonds, and disulfide bridges that maintain theproteins shape.

Some proteins can return to their functionalshape after denaturation, but others cannot,especially in the crowded environment of thecell. Usually denaturation is permanent


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Enzymes

Proteins that catalyze (speed up)chemical reactions without being used upor destroyed in the process.

Anabolic (putting things together) andcatabolic (breaking things down)functions.


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Immune function (antibodies)

Antibodiesare proteins that attack andinactivate bacteria and viruses that causeinfection.


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Basic Principles of Protein Purification


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Basic Principles of Protein Purification

Ammonium sulfate fractionation

Cell OrganelleHomogenization

Macromolecule

Nucleicacid

Carbohydrate (Lipid)

Size Charge Polarity Affinity

Small molecule CellDebrisProtein

Amino acid, Sugar,

Nucleotides, etc

Gel filtration,

SDS-PAGE,

Ultrafiltration

Ion exchange,

Chromatofocusing,Disc-PAGE,

Isoelectric focusing

Reverse phasechromatography,

Salting-out

Affinitychromatography,

Hydroxyapatite


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Protein Determination

Why Quanti fy Protein?

It is often the denominator for presenting

enzyme activity (i.e., mol of activity per

minute per mg protein).Important to know for feeding livestock.


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Biuret Reaction

In alkaline solutions, Cu2+complexes withthe C-N bonds in protein. The result is a

purple color.

This method in relatively insensitive.Tris buffer and other substances often

interfere.Wharton and McCarty, 1980


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The BCA (Bioinchoninic Acid) Method

Uses a similar principle as that described in the biuret

reaction except that BCA is included and sensitivity isincreased.

This process is a two-step reaction.

Protein + Cu2+

+ OH-

Cu1+

Cu1+ + 2 BCA Cu1+/BCA chromophore (562 nm).


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Lowry Method

This method relies upon both the biuret reaction andthe reduction of arsenomolybdate reagent (Folin

reagent) by tryptophan and tyrosine. Consequently,

what type of proteins will give higher absorbances?

Tris buffer and reducing compounds often interfere.


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Coomassie Blue Dye Method (a.k.a.

The Bradford Method)

This method relies on the binding of protein to

Coomassie Brilliant Blue G-250 which causes an

absorbance shift from 465 nm to 595 nm.


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Coomassie Blue Dye Method (a.k.a.

The Bradford Method)

The Coomassie dye binds primarily with basic and

aromatic side chains. The interaction with arginine is

very strong and less strong with histidine, lysine,

tyrosine, tryptophan, and phenylalanine.

About 1.5 to 3 molecules of dye bind per positive

charge on the protein.


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Other Nitrogen Analyses

I norganic Nitrogen

Nitrate - Often determined by converting NO3-

to NO2

-. Ion specific electrodes are available.

Ammonium- Several colorimetric methods are

used. Ion specific electrodes are also an

option. Because of the ammonium/ammonia

equilibrium pH is kept low to prevent

volatilization.


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Crude Protein and Total N

In feeds and other plant tissue, protein is

often calculated from the total N

concentration. To convert total N to

protein, multiply by 6.25. To convert

protein to total N multiply by 0.16.


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The Kjeldahl Method

Tissue is digested in sulfuric acid to

convert nitrogen to ammonium.

Ammonium is converted to ammonia

which leaves the vessel. Ammonia

vapors are then trapped.Ammonia is converted back to

ammonium for colorimetry or titration.


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Proteomics: A Challenge for Technology and

Information Science

What is proteomics?


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What is proteomics?

Proteomics includes not only the

identification and quantification of proteins,

but also the determination of their

localization, modifications, interactions,

activities, and, ultimately, their function.

-Stan Fields in Science, 2001.

Genomics vs Proteomics


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Genomics vs. Proteomics

Similarities:

Large datasets, tools needed for annotation andinterpretation of results

Differences:

Genomics generally mature technologies, dataprocessing methods, questions asked usually involvequantitative changes in RNA transcripts (microarrays)

Proteomics

still evolving, complexity of proteinbiochemical properties: expression changes,modifications,interactions, activities many questions to ask and data tointerpret, methods changing, different approaches (massspec, arrays etc.),

Genomics, Proteomics, and Systems Biology


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Genomics, Proteomics, and Systems Biology

mature

prototype

em

erging

genomic

DNAmRNA

sequencing arrays

genomics

protein

cataloguing

protein

products

functional

protein

quantitative

profiling

protein

phosphorylation

Protein

dynamics

Protein

Modifications

sub cellular

location

catalytic

activity

descriptive protein

interaction maps

3D structure

proteomics

measure

and define

properties

system

identify

system

components

interactions

between

components

computational biology


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Thank you

protein (structures and functions)

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