Aug 26 2011

BT-202Netaji Subhas Institute of Technology,

Dwarka, New Delhi.Dr. Amita Pandey

Aug 26, 2011

Learning check!

Serine has pK values of 2.19 and 9.21. What is its estimated pI?•2.19•5.70•7.00•9.21

By convention amino terminal residue of a peptide chain is written as the right-most residue.•True•False

• The alpha amino groups of all amino acids have a charge at pH 7.0.positive

Which ionic species is most prevalent at the pI.• H2NCH2COOH

• H3N+CH2COOH

• H3N+CH2COO-

• H2NCH2COO-

In which pH range is glycine fully protonated.• Above pH 10.0• pH 2.35 - pH 9.78• pH 2.35 +1/-1• Below pH 1.0

Which side chain is most likely to be negatively charged @ pH 7.0?•Arginine•Aspartic acid•Tyrosine•Cysteine

Which amino acid side chain is NOT aromatic?•Trytophan•Methionine•Tyrosine•Phenylalanine

Structure of proteins

Proteins perform diverse functions in the cell.

Primary Structure

Function of a protein depends upon its AA sequence.-proteins with different functions have different AA sequence.-various genetic disorders have defective proteins.-functionally related proteins have similar AA sequence.

Polymorphic proteins

Same protein with different amino acid composition is called polymorphic protein.

-20%-30% proteins in humans are polymorphic. Eg ABO blood group.

-polymorphism also exists among distantly related species.

First protein Sequence

Frederick Sanger (1953)AA sequence of hormoneinsulin

End Group Analysis• Identify number of terminal AAs– Number of chains/subunits

• Identify specific AA• Sanger method (FDNB)

End Group AnalysisDansyl chloride

•Reacts with primary amines

– N-terminus

•Yields dansylated polypeptides

•Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA

•Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)

Dabsyl chloride

Edman degradation

• Only single AA from the N-terminus is cleaved

• Entire protein sequence can be deduced• Identify using NMR, HPLC, etc.• Sequenator (entire process for proteins <

100 residues)

Sequencing larger proteins

• Formation of smaller segments to assist with sequencing

• Process:– Cleave protein into specific fragments

• Chemically or enzymatically

• Break disulfide bonds

– Purify fragments

– Sequence fragments

– Determine order of fragments and disulfide bonds

Breaking disulfide bond

• performic acid

• Cys residues form

cysteic acid

• Acid can oxidize

other residues, so

not ideal

Breaking disulfide bond

β-2-Mercaptoethanol

(HSCH2CH2OH)

Dithiothreitol (DTT)

(HSCH2CH(OH)CH(OH)CH

2SH)

Enzymatic and Chemical Cleavage

• Enzymatic

–Endopeptidases

• Chemical

-Cyanogen bromide (CNBr)

peptide fragments

Sequencing

Edman procedure

Ordering of peptides

Cleaved again withdifferent enzyme or chemical

Learning check

• A protein is cleaved with cyanogen bromide to yield the following sequences:– Arg-Ala-Tyr-Gly-Asn– Leu-Phe-Met– Asp-Met

• The same protein is cleaved with chymotrypsin to yield the following sequences: – Met-Arg-Ala-Tyr– Asp-Met-Leu-Phe– Gly-Asn

• What is the sequence of the protein?Asp-Met-Leu-Phe-Met-Arg-Ala-Tyr

Protein sequencing

• Mass spectrometry-20-30 AA long fragments.

• Determination of protein sequence from DNA sequence-www.ncbi.nlm.nih.gov

Genome sequencing

organism Size of genome Year

Haemophilus 1.8 Mb 1995

Saccharomyces 12.1 Mb 1996

C. elegans 100Mb 1998

Fruit fly 139 Mb 2005

Human 3.5 Gb 2006

Consensus sequences

Synthesis of peptides

Three ways to obtain a peptide are

-from the tissue-genetic engineering-chemical synthesis

Chemical synthesis

9-fluorenylmethoxycarbonyl

Proteins

• Make up about 15% of the cell• Have many functions in the

cell– Enzymes– Structural– Transport– Motor– Storage– Signaling– Receptors– Gene regulation

Protein folding

• The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions

• Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape

Non-covalent bonds in protein folding

Globular protein

Hydrogen Bonds in Proteins

Protein folding

• Proteins shape is determined by the sequence of the amino acids

• The final shape is called the conformation and has the lowest free energy possible

• Denaturation is the process of unfolding the protein– with heat, pH or chemical compounds

Protein folding

• Renaturation is the process of protein regaining its native conformation

• Eg. Ribonuclease

• Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner

Protein folding

-helix – protein turns like a spiral – fibrous proteins (hair, nails, horns)

-sheet – protein folds back on itself as in a ribbon –globularprotein

Sheets

• Core of many proteins is the sheet

• Form rigid structures with the H-bond

• Can be of 2 types– Anti-parallel – run in

an opposite direction of its neighbor (A)

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

Helix

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

• Usually in proteins that span a membrane

• The helix can either coil to the right or the left

• Can also coil around each other – coiled-coil shape

Protein structure

Domains

• A domaindomain is a basic structural unit of a protein structure – distinct from those that make up the conformations

• Part of protein that can fold into a stable structure independently

• Different domains can impart different functions to proteins

• Proteins can have one to many domains depending on protein size

Domains

Protein Familie

s

• Have similarities in amino acid sequence and 3-D structure

• Have similar functions such as breakdown proteins but do it differently

Proteins – Multiple Peptides

• Non-covalent bonds can form interactions between individual polypeptide chains– Binding site – where proteins interact

with one another– Subunit – each polypeptide chain of

large protein– Dimer – protein made of 2 subunits• Can be same subunit or different subunits

Single Subunit Proteins

Different Subunit Proteins

• Hemoglobin–2 globin subunits–2 globin subunits

Protein Assemblies

• Proteins can form very large assemblies

• Can form long chains if the protein has 2 binding sites – link together as a helix or a ring

• Actin fibers in muscles and cytoskeleton – is made from thousands of actin molecules as a helical fiber

Types of Proteins

• Globular ProteinsGlobular Proteins – most of what we have dealt with so far– Compact shape like a ball with

irregular surfaces– Enzymes are globular

• Fibrous ProteinsFibrous Proteins – usually span a long distance in the cell– 3-D structure is usually long and rod

shaped

Important Fibrous Proteins

• Intermediate filaments of the cytoskeleton – Structural scaffold inside the cell• Keratin in hair, horns and nails

• Extracellular matrix – Bind cells together to make tissues– Secreted from cells and assemble in

long fibers • Collagen – fiber with a glycine every third

amino acid in the protein• Elastin – unstructured fibers that gives

tissue an elastic characteristic

Collagen and Elastin

Stabilizing Cross-Links

• Cross linkages can be between 2 parts of a protein or between 2 subunits

• Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid cysteine

Proteins at Work

• The conformation of a protein gives it a unique function

• Ligand – the molecule that a protein can bind

• Binding site – part of the protein that interacts with the ligand– Consists of a cavity formed by a specific

arrangement of amino acids

Ligand Binding

Formation of Binding Site

• The binding site forms when amino acids from within the protein come together in the folding

• The remaining sequences may play a role in regulating the protein’s activity

Antibody Family

• A family of proteins that can be created to bind to almost any molecule

• AntibodiesAntibodies (immunoglobulins) are made in response to a foreign molecule ie. bacteria, virus, pollen… called the antigenantigen

• Bind together tightly and therefore inactivates the antigen or marks it for destruction

Antibodies

• Y-shaped molecules with 2 binding sites at the upper ends of the Y

• The loops of polypeptides on the end of the binding site are what imparts the recognition of the antigen

• Changes in the sequence of the loops make the antibody recognize different antigens - specificity

Antibodies

Binding Strength• Can be measured directly• Antibodies and antigens are mixing

around in a solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules

• This process continues until the equilibrium equilibrium is reached – number sticking is constant and number leaving is constant

• This can be determined for any protein and its ligandligand

Equilibrium

Constant

• Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium constant (K)equilibrium constant (K)

• Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together

Enzymes as Catalysts

• Enzymes are proteins that bind to their ligand as the 1st step in a process

• An enzyme’s ligand is called a substratesubstrate– May be 1 or more molecules

• Output of the reaction is called the product

• Enzymes can repeat these steps many times and rapidly, called catalysts

Enzymes at Work• Lysozyme is an important enzyme that

protects us from bacteria by making holes in the bacterial cell wall and causing it to break

• Lysozyme adds H2O to the glycosidic bond in the cell wall

• Lysozyme holds the polysaccharide in a position that allows the H2O to break the bond – this is the transition state transition state – state between substrate and product

• Active siteActive site is a special binding site in enzymes where the chemical reaction takes place

Lysozyme

• Non-covalent bonds hold the polysaccharide in the active site until the reaction occurs

Features of Enzyme Catalysis