Understanding Proteins

Quiz 1:
Date: 9th February, Wednesday, 2011. Time: 8 AM – 8.50 AM Venue: LIST of rooms with Roll numbers provided in the website of the course. If your roll number is not included in this list, inform the instructor BEFORE 7th February, 2011. Any change in this list will not be entertained after 7th February. Syllabus: All the topics discussed in BT101 class, till 7th February 2011. Note: 1) You MUST carry your scientific calculator. 2) All questions will be of MULTIPLE CHOICE TYPE. See the MODEL QUESTIONS given at the website 3) Do not use pencil to write answers, 4) Do carry your institutional ID card, 5) Mobile phones will not be allowed in the examination hall. http://shilloi.iitg.ernet.in/~biotech/BT%20101%20Modern%20Biology.htm

Reminders

• Proteins are polymer made up of Amino Acids. • There are 20 amino acids • Functions of Proteins: Enzyme, Ion channels, Signaling, Mechanical structure Common structure of amino acids:

R is different in different amino acids

R is called “Side Chain”

Different Types of Side Chains
Non-polar, Aliphatic Non-polar, Aromatic

Different Types of Side Chains
Polar Uncharged Polar +ve charged Polar –ve charged

Ionization of Amino Acids

Zwitterion Low pH High pH

Titration Curve of Glycine

Each Amino Acids have 2 or more pKa pI = Isoelectric point = pH at which net charge is zero A protein made up of large number of amino acids also have a unique pI at which net charge on the protein is zero.

Titration Curve of Glutamic acid It has 3 different pKa

Proteins are Polypeptide
Proteins are formed by polymerization of amino acids by formation of Peptide Bonds

Peptide bond

Proteins are Polypeptide

Example of a pentapeptide

Proteins are large peptide made up of 100s of amino acids

A Polypeptide Is Not Planner
Peptide bond is partially rigid and remain in a plane. Other atoms remain projected from that plane

Polypeptide (or Protein) Folds in 3D

Linear poly-Peptide

Folded Poly-peptide (Native structure of protein)

In water, hydrophobic residues (shown as black circles) tend to hide from water, there by spontaneously folding protein to its native structure.

A protein is functional only in its native structure Chemicals like, urea, GdnHCl, and Heat, unfold protein. These are called denaturing agents. Extent of unfolding can be measured by determining residual function of the protein Tm = Melting point. At this point the protein is 50% unfolded

Anfinsen’s Experiment
• • Sequence determines structures Proof : renaturation of ribonuclease by Anfinsen after denaturation – Correct disulfide bonds

Transition state

G Unfolded state ∆G = 5-15 kcal/mol Folded state

Reaction process

Thermodynamic of Protein Folding
Unfolded protein has Higher Entropy ∆G = ∆H – T∆S In this case, ∆S < 0; to make ∆G < 0, ∆H must be Negative Interaction between atoms of the protein itself Interaction between atoms of protein and atoms of water. Non-covalent interaction : H-bond, ionic, VdW. Lots of such interactions A large negative ∆H ∆G is Negative (usually – 5 to -15 kcal/mol) Folded protein have lower entropy.

Folding of the protein disturbs organized structure of water, increasing entropy of water. So, net entropy of the system (Protein + Water) increases on protein folding

Understanding Proteins

1° 2°,and 3°Protein Structure ,

(local structure)

(A folded protein)

(made up of two or more similar or dissimilar proteins)

Non-functional Functional

Important Secondary Structures

Alpha Helix Chain twists into a helix with 36 residues per 10 turns

Important Secondary Structures

Beta-Pleated Sheet

Molecular Representation

ATP

Ball-and-stick Ball for atom and stick for bond Size and color of a ball depend upon atom

Space filling Two ball can fuse if they are bonded

Molecular Representation
A protein usually have thousands of atoms. Ball-and-stick or Space-filling representation are not suitable to show a protein

Space-filling model

Molecular Representation
Ribbon Diagram

Helix

N terminal C terminal

β strand

Very good to understand the fold architecture of a protein. Note the type of ribbon for different secondary structure and meaning of arrow head.

Molecular Representation

Ribbon (Cartoon) Ribbon Diagram is better for representation of a protein

Molecular Representation

Molecular Surface Suitable to: Understand charges on surface; Detect active site/pockets

Protein Chemistry of Human Hair
•Main constituent of hair : α keratin •It’s a helical protein. •Two copies coil over each other. Forms intermolecular disulphide bonds •Further coiling leads to thick fibrous structure.

Protein Chemistry of Human Hair
Curling of Hair:

Human Hemoglobin

Heme Gr.

Adult Hemoglobin •Made up of two identical subunit α and β, each present in two copies •Heme Gr. binds oxygen

Sickle-cell Disease
A single Base Mutation:

Hydrophilic

Hydrophobic

Sickle-cell Disease
•Glu Val mutation generates a hydrophobic patch in each β subunit. •In deoxygenated state such hydrophobic patches get exposed. •Two hemoglobin interacts through those hydrophobic patch and stick.

•Forms elongated strands of sticking hemoglobins •Crystallize and precipitates

•RBC changes shape •Oxygen carrying capacity decrease •Sickle shaped RBC blocks blood vessels •Increased lysis of RBC Sickle cell Normal

Problems Due to Misfolding of Proteins
• In normal conditions, proteins fold in the cytosol, as each molecule is produced. • Some time, protein folding is assisted; Molecular machines made up of proteins help to fold proteins; Those machines are called Chaperones. • Misfolding of protein leads to loss of function of the protein and aggregation. • Such aggregation lead to diseases like: Alzheimer’s, Mad cow,CreutzfeldtJakob Alzheimer’s APP protein gets chewed to create small peptide Amyloid beta (Aβ42). Amyloid beta forms aggregates (shown in panel b) This leads to malfunction of brain cells.

Problems Due to Misfolding of Proteins
Prion Disease

•PrPC is normal protein. •PrPSc is the isoform of PrPC •PrPSc fold differently and tends to form aggregates •PrPSc induces conversion of PrPC to PrPSc •PrPSc is infectious

Deciphering Protein Sequence and Structure
Sequence Identification: •Edman degradation: Step by step reaction to identify amino acids •Digest the protein small peptides Mass Spectroscopy (based on m/z ratio) •DNA RNA Protein •If you know the DNA sequence, you decipher the Protein sequence •DNA sequence is comparatively easier than protein sequencing

Structure Determination: •X-ray crystallography •NMR •Theoretical models

X-Ray Crystallography
Get a protein crystal Shine with X-ray
~0.5mm

Fit a model of the protein to electron density map. Refine the model

Get diffraction pattern Create electron density map

Predicting Protein Structure
Given: Amino acid sequence of a protein Predict the 3D structure when it is folded in water

Theoretical folding of protein to predict structure:

•Fold in all possible orientation and calculate free energy of each structure. •Identify structure with lowest free energy •Huge number of combinations. •Can not be solved by existing computing schemes What is possible? Rough and iterative algorithms which are faster and doable

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