Proteins

ProteinsDr. Deepak K. Gupta

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Syllabus

• Biological importance

• Amino acids : Classification.

• Introduction to peptides.

• Proteins - Classification : simple and conjugated; Globular and fibrous.

• Charge properties - Buffer action.

• Denaturation


Introduction : Protein

• Most abundant organic molecules of the living system

• Its fundamental basis of structures and function of life.

• 50 % of dry weight of every cell

• It’s a polymer of L α-amino acids.

• 300 different amino acids occur in nature –only 20 as standard amino acids.

• 21st amino acid added - Selenocysteinewww.facebook.com/notesdental

BIOMEDICAL IMPORTANCE• Beside forming long chain polypeptide unit of

protein, amino acids have additional functions– nerve transmission

– biosynthesis of porphyrins, purines, pyrimidines,and urea

– Short polymers of AA – peptides

– Neuroendocrine system - hormones, hormone releasing factors, neuromodulators, or neurotransmitters

– Microorganisms : D- and L-α-amino acids• Therapeutic value: antibiotics bacitracin and gramicidin A and

the antitumor agent bleomycin

• Some may be toxic


Amino Acid• It’s a group of organic

compounds containing two functional groups – amino (-NH2) and carboxyl group (-COOH)

• Its also called Zwitter Ion–both acidic and basic functional group (dipolar ion)

• This property is known as amphoteric and are often called ampholytes

• Neither humans nor any other higher animals can synthesize 10 of the 20 common amino acids –Essential Amino acids


Classification

• Amino acid has been classified under various ways– Structure

• With side chain containing Aliphatic Side Chains• With Side Chains Containing Hydroxylic (OH) Groups• With Side Chains Containing Sulfur Atoms• With Side Chains Containing Acidic Groups or Their Amides• With Side Chains Containing Basic Groups• Containing Aromatic Rings• Imino Acid

– Polarity• Non Polar• Polar

– Nutritional• Essential and Non-essential


Side chain containing Aliphatic Side Chains

• Simplest amino acids

• Contains branched chain of hydrocarbons


Side Chains Containing Hydroxylic(OH) Groups


Side Chains Containing Sulfur Atoms


Side Chains Containing Acidic Groups or Their Amides


Side Chains Containing Basic Groups


Containing Aromatic Rings


Imino Acid


Classification : Polarity

• Non- polar group : No charge on R group. Ex: Alanine, leucine. Isoleucine, valine, methionine, phenylalanine, tryptophan and proline

• Polar group– No charge on R : no charge on R but posses group

such as hydroxyl, sulfhydryl and amide. Ex: Glycine, serine, threonine, cysteine, glutamine, asparigine and tyrsoine

– Positive R- Lysine, arginine, and histidine

– Negative R – asparatic acid and glutamic acid


Essential Amino Acid (EAA)

• It cant be synthesized in the body and therefore need to be supplied through diet

• Proper growth and maintenance of the individual• Ex. Arginine, Valine, Histidine, Isoleucine, leucine,

lysine, Methionine, Phenylalanine, Threonine, Tryphtophan

• Mnemonics : AV hill, MP TT• Semi-essential amino acid: Adults can synthesize

2 amino acid and not by growing children. Ex: Arginine and histidine

• So in all 8 are essential and 2 semi essential


PEPTIDES• Two AA covalently joined

through a substituted amide linkage – peptide bond

• Dehydration – removal of H2O– OH- Carboxyl group of one

AA

– H+ from amino group of another AA

• Example of a condensation reaction – common biological reactions


POLYPEPTIDES

• Two AA reacts to form dipeptides, Three AA can be joined by two peptide bonds to form a tripeptideand so on.

• Oligopeptide: When a few AA are joined by various peptide linkage

• When many amino acids are joined, the product is called a polypeptide.

• Proteins may have thousands of amino acidresidues Tetrapeptide


Structures of Protein

• Proteins catalyze metabolic reactions, power cellular motion, and forms structural integrity to hair, bones, tendons and teeth

• Human proteins therefore reflects the sophistication and diversity of their biologic roles

• Therefore maturation of a newly synthesized polypeptide into a biologically functional protein

– Requires folding into a specific three-dimensional arrangement, or conformation


Structures of Protein

• During maturation, posttranslational modifications may add new chemical groups or remove it transiently

• Genetic or nutritional deficiencies that impede protein maturation are deleteriousto health.

• Ex: Creutzfeldt Jakob disease, Scrapie, Alzheimer’s disease, and bovine spongiform encephalopathy

• Scurvy - nutritional deficiency that impairs protein maturation.


FOUR ORDERS OF PROTEIN STRUCTURE

• The modular nature of protein synthesis and folding are embodied in the concept of orders of protein structure:– Primary structure: linking amino acid residues in a

polypeptide chain

– Secondary structure: stable arrangements of amino acid residues giving rise to recurring structural patterns into geometrically ordered units; twisting resulting in α-helix or pleated

– Tertiary structure: the three-dimensional assembly of secondary structural units to form larger functional units

– Quaternary structure: It’s the arrangement in space of protein having two or more polypeptide subunits


FOUR ORDERS OF PROTEIN STRUCTURE


PRIMARY STRUCTURE• Primary (1°) structure• Each protein has a

distinctive number and sequence of amino acid residues

• These determines how it folds up into a unique three-dimensional structure

• This in turn determines the function ofthe protein

Bovine Pancreatic Ribonuclease Awww.facebook.com/notesdental

SECONDARY STRUCTURE• 2° structures• Polypeptide chain can arrange

itself into characteristic helical or pleated segments– Given by Pauling and Corey– hydrogen bonding interactions

between adjacent amino acid residues

• Free rotation is possible about only two of the three covalent bonds of the polypeptide backbone• α-carbon (Cα) to the carbonyl carbon (Co)

bond• Cα to nitrogen bond

Psi (Ψ) anglePhi (Φ) angle


SECONDARY STRUCTURE : Alpha Helix

• α helix is twisted byan equal amount about each α-carbon

• With a phi angle of approx. −570

and a psi angle of approx − 470

• Complete turn of the helix contains an average of 3.6 aminoacyl residues

• Distance it rises per turn (pitch) is 0.54 nm

• R groups of each aminoacylresidue in an α helix face outward


View down the axis of an α helix


SECONDARY STRUCTURE : Alpha Helix

• Stability of an α helix arises primarily from hydrogen bonds

• Between the oxygen of carbonyl and the hydrogenatom of nitrogen of the 4th

residue down the polypeptide chain

• Supplemented by van derWaals interactions


SECONDARY STRUCTURE : Beta Sheet

• Extended conformation of polypeptide chains

• Viewed edge-on, form a zigzag or pleated pattern in which the R groups of adjacent residues point in opposite directions

• Stability from hydrogen bonds between the carbonyl oxygens and amide hydrogens of peptide bonds - adjacent segments of β sheet


SECONDARY STRUCTURE : Beta Sheet

• Parallel:polypeptide chain proceed in the same direction amino to carboxyl

• Antiparallel: they proceed in opposite directions


Loops & Bends

• Short segments of amino acids that join two units of secondary structure – 3-4 units

• Globular proteins - compact folded structure

• Nearly one-third of the amino acid residues are in turns or loops where the polypeptide chain reverses direction

• The structure is a 1800 turn involving four amino acid residues


Loops & Bends• Carbonyl oxygen of the

first residue forms a hydrogen bond with the amino-group hydrogen of the fourth residue

• The peptide groups of the central two residues do not participate in any hydrogen bonding

• Gly (small and flexible) and Pro (readily assume the cisconfiguration) residues often occur in turns


Loops & Bends

• Generally found on the surface of a protein

• γ -turn - less common is the, a three residue turn with a hydrogen bond between the first and third residues.


Tertiary Structure

• Entire 3-dimensional conformation of a polypeptide

• Beside H bond, sulfide bond (-S-S), ionic interaction and hydrophobic bond

• Consists of helices, sheets, bends, turns, and loops— assemble to form domains

• Domain is a section of protein structure - perform a particular chemical or physical task– binding of a substrate or other ligand– anchor a protein to a membrane – interact with a regulatory molecules


Single domain:triose phosphate isomerase

• Enzyme triose phosphate isomerase complexedwith the substrate analog 2-phosphoglycerate(red)

• Elegant and symmetrical arrangement of alternating β sheets (light blue) and a helices (green), with the β sheets forming a β-barrel core surrounded bythe helices


Tertiary Structure• Single domain - triose phosphate isomerase, myoglobin• Two domains - lactate dehydrogenase, quinone

oxidoreductase• A polypeptides with 200 amino acids normally consists of

two or more domains

Tetrameric enzyme lactate dehydrogenasewith the substrates NaDh (red) and pyruvate (blue) bound


Quaternary Structures

• Majority of proteins are composed of single polypeptide chains

• Some of protein consists of 2 or more polypeptide chain which may be identical or different

• Such protein are termed as oligomers and poses quaternary structures.

• When it consists of 2 polypeptides - dimers• Homodimers contain two copies of the same

polypeptide chain, while in a heterodimer the polypeptides differ


Properties of Protein

• Solublity: forms colloidal solution instead of true solutions in water – large size of protein

• Molecular Weight: depends on number of amino acid

• Shape: there is wide variety in shape –globular(insulin), oval(albumin), fibrous or elongated (fibrinogen)

• Acidic and basic: depends on ratio of (lysine + arginine) : (Glut + Asp). Ratios greater than 1 is basic and vice-versa


Properties of Protein : Charge

• Protein are isoelectric• Nature of amino acids determines the pH of a

protein• Acidic amino acid (Asp, Glu) and basic amino acid

(His, lys, Arg) – determines the charge on protein• At isoelectric pH, the protein exist as Zwitter-ions

and dipolar ions– Electrically neutral– Minimum solubility– Maximum precipitability– Least buffering capacity


Classification• Proteins are classified on the basis of

– Chemical nature and solubility• Simple• Conjugates• Derived

– Function• Structural• Enzyme or catalytic• Transport• Hormonal• Contractile• Storage• Genetic• Defense• Receptor

– Nutritional Importance• Complete• Partially incomplete• Incomplete


Chemical nature and solubility :

Simple

• They are composed of only amino acid residues

• They are again classified as– Globular Protein : spherical or oval in shape,

soluble in water or other solvent and digestible• Globulin: soluble in neutral and salt solution. Ex: serum

globulin

• Albumin: soluble in water and dilute salt solutions and cogulated by heat. Ex: serum albumin, ova albumin, lactalbumin


Chemical nature and solubility : Simple

• Globular Protein (Cont)…

– Glutelins : soluble in dilute acids, alkalies and mostly found in plants. Ex: Glutelin (wheat), oryzenin (rice)

– Prolamines: soluble in alcohol. Ex: gliadin(wheat), zein (maize)

– Histones: strongly basic proteins, soluble in water and dilute acids but insoluble in dilute ammonium hydroxide. Ex: thymus histone


Chemical nature and solubility : Simple

• Fibrous Protein: fiber like in shape, insoluble in water and resistant to digestion. It again of 3 types

– Collagen: connective tissue protein lacking tryptophan. On heating with boiling water or acids it turns to soluble gelatin

– Elastin: elastic tissues such as tendons and ateries

– Keratin: present in the exoskeleton structures. Ex: hair, nails, horns


Chemical nature and solubility : Conjugate Protein

• Beside amino acid, it contains a non-protein moiety known as prosthetic group or conjugating group. Its again of 6 types– Nucleoprotein: nucleic acid (DNA or RNA)

– Glycoprotein: prosthetic group is carbohydrate which is less than 4 % and when it exceeds 4% its called mucoprotein. Ex: mucin (saliva), ovamucid(egg white)

– Lipoprotein: found in the conjugation with lipids. Ex: serum lipoprotein, membrane lipoprotein


Chemical nature and solubility : Conjugate Protein

• Phosphoprotein: phosphoric acid as conjugate. Ex: casein(milk), vitelline (egg yolk)

• Chromoprotein: prosthetic group is colored in nature. Ex: Hemoglobins, cytochromes

• Metalloprotein: it contains metal ions such as Fe, Co, Zn, Cu, Mg,


Chemical nature and solubility : Derived Protein

• Denatured or degraded product of simple or conjugated protein

• Its of 2 types– Primary derived protein: denatured or cogulated or

first hydrolyzed product of proteins. They are• Cogulated proteins: denatured protein produced by agents

such as heat, acids, alkalies• Proteans: earliest product of protein hydrolysis by enzymes,

dilute acids, alkalies etc. Insoluble in water• Metaprotein: second stage of protein hydrolysis obtained by

treatment with slightly stronger acids and alkalies

– Secondary derived protein: progressive hydrolytic product of protein hydrolysis. Ex: proteoses, peptones, polypeptides and peptides


Denaturation

• The phenomenon of disorganization of native protein structure

• It results in the loss of secondary, tertiary and quaternary structure of proteins.

• It involves the change of physical, chemical and biological properties

• Agents of Denaturation– Physical agents: Heat, UV radiation, X-rays and violent

shaking (centrifuge)– Chemical Agents: Acids, alkalies, organic solvents

(ether, alcohol), salts of heavy metals, urea, salicylate


Denaturation

• Primary structures remains intact i.e peptide linkage are not broken

• Loses its biological activity

• Insoluble in solvent which was previously soluble

• Viscosity increases while its surface tension decreases

• Its more easily digestible

• Its usually irreversible, but careful denaturation(renaturation) is reversible. Ex: Hemoglobin is renatured on removal of salicylates


Denaturation

• Coagulation

– Irreversible denaturation of protein to semi-solid viscous precipitate

– Albumins and globulins – coagulable proteins

• Flocculation

– Protein precipitation at isoelectric pH.

– Precipitate is known as flocculum

– Casein – milk protein, prepared by adjusting isoelectric pH by dilute acetic acid

– Its reversible, but on heating it turns to be irreversible


Refrences

• Harper's Illustrated Biochemistry, 30E (2015)

• Biochemistry - U. Satyanarayan and U. Chakrapani3rd edition

• Lehninger Principles of Biochemistry, Fourth Edition - David L. Nelson, Michael M. Cox

• Biochemistry - Garrett And Grisham 2nd Ed 1998

• Biochemistry Stryer 5th Edition repost

• Color Atlas of Biochemistry 2005


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Proteins

Health & Medicine