AMINO ACIDS &

PROTEINS

Centre for Nano science and TechnologyCourse: Introduction to Nano technology.Code: NST 611Course instructor: Dr. A. Kasi Viswanath.

PRESENTED BY

ROOPAVATH UDAY KIRANM.Tech 1st year

Overview of the Presentation

AMINO ACIDS

TYPES OF AMINO ACIDS

PROPERTIES OF AMINO ACIDS

PROTEINS

STRUCTURE OF PROTEINS

PROPERTIES

PROTEIN SYNTHESIS

PROTEINS AND AMINO ACIDS IN NANO SCIENCE AND TECHNOLOGY

AMINO ACIDS Amino acids are biologically important organic compounds

made from amine (-NH2) and carboxylic acid (-COOH)functional groups, along with a side-chain specific to eachamino acid. The key elements of an amino acid are carbon,hydrogen, oxygen, and nitrogen, though other elements arefound in the side-chains of certain amino acids.

About 500 amino acids are known and can be classified inmany ways.

In the form of proteins, amino acids comprise the secondlargest component (after water) of human muscles, cells andother tissues. Outside proteins, amino acids perform criticalroles in processes such as neurotransmitter transport andbiosynthesis.

•Amine group acts like a base, tends to be positive.• Carboxyl group acts like an acid, tends to be negative.• “R” group is variable, from 1 atom to 20.• Two amino acids join together to form a dipeptide. • Adjacent carboxyl and amino groups bond

together.

Amino acids having both the amine andcarboxylic acid groups attached to the first(alpha-) carbon atom have particular importancein biochemistry.

They are known as 2-, alpha-, or α-amino acids(generic formula H2NCHRCOOH in most caseswhere R is an organic substituent known as a"side-chain");often the term "amino acid" is usedto refer specifically to these.

They include the 22 proteinogenic ("protein-building") amino acids which combine intopeptide chains ("polypeptides") to form thebuilding blocks of a vast array of proteins.

Twenty of the proteinogenic amino acids are encoded directly bytriplet codons in the genetic code and are known as "standard"amino acids.

The other two ("non-standard" or "non-canonical") are pyrrolysine(found in methanogenic organisms and other eukaryotes) andselenocysteine (present in many noneukaryotes as well as mosteukaryotes). For example, 25 human proteins includeselenocysteine (Sec) in their primary structure, and the structurallycharacterized enzymes (selenoenzymes) employ Sec as the Aminoacid 2 catalytic moiety in their active sites.

Pyrrolysine and selenocysteine are encoded via variant codons;for example, selenocysteine is encoded by stop codon and SECISelement. Codon–tRNA combinations not found in nature can alsobe used to "expand" the genetic code and create novel proteinsknown as alloproteins incorporating non-proteinogenic aminoacids. Many important proteinogenic and non-proteinogenicamino acids also play critical non-protein roles.

Cysteine

• The AA Cysteine exists as a dimer:

cysteine

COOHC

NH2

CH2

H

HS[O]

[H]2

HCOO C

NH2

CH2

H

S COOHC

NH2

CH2

H

S

cystine

a disulfide linkage

Zwitterions• An acid -COOH and

an amine -NH2 groupcannot coexist

• The H+ migrates to the-NH2 group

• COO- and NH3+ are

actually present, calleda “Zwitter ion”

• Zwitter ion = compound where both a positive charge and a negativecharge exist on the same molecule

• AA are ionic compounds• They are internal salts• In solution their form changes

depending on the pH

Formation of a Dipeptide

Dehydration synthesis

Amino Acid + Amino Acid --> Dipeptide

Amino Acid + Dipeptide --> Tripeptide

A.A. + A.A. + …..+ Tripeptide --> Polypeptide

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

peptide bonds peptide bonds

side chains

amino acidresidues

Polypeptides

PROTEINS Proteins are large biological molecules consisting of

one or more chains of amino acids.

Proteins perform a vast array of functions withinliving organisms, like:

Catalyzing metabolic reactions

Replicating DNA

Responding to stimuli and

Transporting molecules from one location to another.

Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors.

• Many proteins are enzymes that catalyzebiochemical reactions and are vital to metabolism.

• Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape.

• Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle.

Proteins -

• Proteins make up all living materials

Characteristics of protein:

• Contain carbon, hydrogen, oxygen, nitrogen, and sulfur

• Serve as structural components of animals

• Serve as control molecules (enzymes)• Serve as transport and messenger

molecules• Basic building block is the amino acid

•Functions of proteins:

1. Help fight disease

2. Build new body tissue

3. Enzymes used for digestion and otherchemical reactions are proteins

(Enzymes speed up the rate of a reaction)

4. Component of all cell membranes

PURIFICATION

Proteins may be purified from other cellular components using a variety of techniques such as:

• Ultracentrifugation

• Precipitation

• Electrophoresis

• Chromatography

Methods commonly used to study protein structure and function include:

• Immunohistochemistry

• Site-directed mutagenesis

• Nuclear magnetic resonance

• Mass spectrometry.

Proteins are linear polymers of amino acids

R1

NH3＋ C CO

H

R2

NH C CO

H

R3

NH C CO

H

R2

NH3＋ C COOー

H

＋

R1

NH3＋ C COOー

H

＋

H2OH2O

Peptide bond

Peptide bond

The amino acid sequence is called as

primary structureA A

FNG

GS

T

S

DK

A carboxylic acid condenses with an amino group with the release of a water molecule

Amino acid sequence is encoded by DNA base sequence in a gene

・CGCGAATTCGCG・

・GCGCTTAAGCGC・

DNA molecule

＝

DNA base sequence

Gene is protein’s blueprint, genome is life’s blueprint

Gene

GenomeDNA

Protein

Gene Gene

Gene

Gene

GeneGene

GeneGene

GeneGeneGeneGene

GeneGene

Protein Protein

ProteinProtein

Protein

ProteinProtein

Protein

Protein

Protein

Protein

Protein

Protein

Protein

Protein Synthesis

BIOSYNTHESIS

Making a Protein—Transcription

• First Step: Copying of genetic information from DNA to RNA is called Transcription.

Why? DNA has the genetic code for the protein that needs to be made, but proteins are made by the ribosomes—ribosomes are outside the nucleus in the cytoplasm.

DNA is too large to leave the nucleus (double stranded), but RNA can leave the nucleus (single stranded).

• Part of DNA temporarily unzips and is used as atemplate to assemble complementary nucleotidesinto messenger RNA (mRNA).

• mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome.

• mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome.

Making a Protein—Translation

• Second Step: Decoding of mRNA into a protein is calledTranslation.

• Transfer RNA (tRNA) carries amino acids from thecytoplasm to the ribosome.

These amino acids come from the food we eat. Proteinswe eat are broken down into individual amino acids andthen simply rearranged into new proteins according to theneeds and directions of our DNA.

• A series of three adjacent bases inan mRNA molecule codes for aspecific amino acid—called a codon.

• A triplet of nucleotides in tRNA thatis complementary to the codon inmRNA—called an anticodon.

• Each tRNA codes for a differentamino acid.

Amino acid

Anticodon

• mRNA carrying the DNA instructions and tRNA carrying amino acids meet in the ribosomes.

• Amino acids are joined together to make a protein.

Polypeptide = Protein

Chemical synthesis

• Short proteins can also be synthesized chemically by a familyof methods known as peptide synthesis.

• Chemical ligation to produce peptides in high yield.

• Chemical synthesis allows for the introduction of non-naturalamino acids into polypeptide chains, such as attachment offluorescent probes to amino acid side chains.

• These methods are useful in laboratory biochemistry andcell biology, though generally not for commercialapplications. Chemical synthesis is inefficient for polypeptideslonger than about 300 amino acids.

• Synthesized proteins may not readily assume their nativetertiary structure.

• Most chemical synthesis methods proceed from C-terminusto N-terminus, opposite the biological reaction.

Protein Structure

• Primary Structure

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

CCHN

R

OH

AA 1 AA 2 AA 3 AA 4 AA 5 AA 6

With any 6 of the 20 common AA residues, the number of possible combinations is 20 x 20 x 20 x 20 x 20 x 20 = 64,000,000

(and this is not nearly large enough to be a protein!)

• Primary Structure

• A typical protein could have 60

AA residues. This would have

2060 possible primary

sequences.

2060 = 1078

This results in more possibilities

for this small protein than there

are atoms in the universe!

• Sometimes small changes in the

1o structure do not alter the

biological function, sometimes

they do.

Each Protein has a unique structure

Amino acid sequence

NLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG

Folding!

Basic structural units of proteins: Secondary structure

α-helix β-sheet

Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.

Secondary Structure Repeating patterns

within a region Common patterns

helix pleated sheet

Originally proposed byo Linus Paulingo Robert Corey

helix Single protein chain Shape maintained by

intramolecular H bondingbetween -C=O and H-N-

Helical shapeo helix is clockwise

pleated sheet Several protein chains Shape maintained by

intramolecular H bondingand other attractive forces between chains

Chains run anti-paralleland make U turns at ends

Random Coils Few proteins have

exclusively helix or pleated sheet

Many have non-repeatingsections called:Random Coils

Tertiary Structure:

The Three dimensional arrangement of every atom in the molecule

Includes not just the peptide backbone but the side chains as well

These interactions are responsible for the overall folding of the protein

This folding defies its function and it’s reactivity

The Tertiary structure is formed by the following

interactions:

– Covalent Bonds

– Hydrogen Bonding

– Salt Bridges

– Hydrophobic Interactions

– Metal Ion Coordination

Tertiary Structure

Quaternary Structure:

– Highest level of organization

– Determines how subunit fit together

– Example Hemoglobin (4 sub chains)• 2 chains 141 AA

• 2 chains 146 AA

- Example - Collagen

Hierarchical nature of protein structure

Primary structure (Amino acid sequence)

↓

Secondary structure （α-helix, β-sheet）

↓

Tertiary structure （Three-dimensional structure formed by assembly of secondary structures）

↓

Quaternary structure （Structure formed by more than one polypeptide chains）

Close relationship between protein structure and its function

enzyme A

B

A

Binding to A

Digestion of A!

enzyme

Matching the shape to A

Hormone receptor AntibodyExample of enzyme reaction

enzyme

substrates

Denaturation Denaturation

Any physical or chemical agent that destroys the conformation of a protein is said to “denature” it

Examples:

Heat (boil an egg) to gelatin

Addition of 6M Urea (breaks H bonds)

Detergents (surface-active agents)

Reducing agents (break -S-S- bonds)

Acids/Bases/Salts (affect salt bridges)

Heavy metal ions (Hg2+, Pb2+)

Some denaturation is reversible

Urea (6M) then add to H2O

Some is irreversible

Hard boiling an egg

Summary

Proteins are key players in our living systems.

Proteins are polymers consisting of 20 kinds of aminoacids.

Each protein folds into a unique three-dimensionalstructure defined by its amino acid sequence.

Protein structure has a hierarchical nature.

Protein structure is closely related to its function.

Protein structure prediction is a grand challenge ofcomputational biology.

IMPORTANCE OF PROTEINS IN NANO SCIENCE

Protein Bio Devices

Nano Drug delivery

Drug designing

Protein based Bio materials

Self assembly, Etc.,

Gastrin Releasing Protein (GRP)

Receptor Specific Gold Nano-rods

General structure of GNR-BBN conjugates.

GNR-BBN conjugates have

very high binding affinity

toward GRP receptors in

cancer cells and can easily

enter into the cancer cell.

The selective delivery of

GNRs to tumoral region can

be achieved by attaching a

target-specific protein

(BBN).

Bombesin (BBN)

Gold nanorod (GNR)

Fluorescently-tagged Proteins Combination of molecular and cell biological

studies analyze in vivo localization of proteins expressed with a fluorescent “tag”

Important that “tag” does not interfere with protein activity

Can examine localization of proteins containing different fluorophores

Self-assembly:

• Peptide of 16 AA

• Alternating polar/nonpolar

• Form stable β-strands and β-sheets

• Form nanofibers by hydrophobicity

• Matrices with high H2O content

Self-assembly:

• Charged head group and nonpolar tail

• Form nanotubes and nanovesicles

• Form interconnected network

• Similar to carbon nanotube behavior

Self-assembly:

Non-fibrous ECM components

• Adhesion proteins

• Growth factors

• Topography

Protein Engineering In all cells proteins have:

Enzyme activities

Structural roles

Allows detailed in vitro studies

Proteins can also be made to do useful operations both in vitro and in cells

Protein engineering involves processes that modify or improve proteins

Protein Engineering manipulates protein production, incorporating modifications to “improve” proteins

Recombinant proteins can provide much information about protein function both in vitro and in vivo

Engineered proteins have huge potentials in biotechnology and medicine

Improving Proteins• Quite difficult to improve on activities

of proteins for any particular cell –evolution is very efficient!

• Can replace mutated (dysfunctional) proteins

• Recent advances have tried to make use of novel or uncommon amino acids

- Selenocysteine: in a few proteins in all cells (e.g. formatedehydrogenase in bacteria, glutathione peroxidase in mammals)

- Pyrrolysine: found in methanogenic group of Archaea

Uncommon Amino Acids

• Expansion of genetic code to uncommon amino acids requires several changes in cells:

- Specific aminoacyl-tRNA synthetase

- Specific tRNA

- New metabolic pathways (??) for synthesis of above molecules

• Scientists have used similar approaches to incorporate unnatural amino acids

• Added one at a time, but over 30 different amino acids have been introduced

• Performed for E. coli, yeast, mammalian cells

(a) ketone; (b) azide; (c) photocrosslinker; (d) highly fluorescent; (e) heavy atom for use in crystallography; (f) long-chain cysteine analogue