PROTEIN STRUCTURE AND FUNCTION

Proteins Are Where It’s At

• Proteomics• Gene regulation• Drug Discovery• Understanding evolution• Etc.

Proteins are Where It’s Been

• Enzymes ß-galactosidase

• Antibodies Anti-Hepatitis B

• Hormones Human Growth Hormone Estrogen Testosterone

Proteins are Where It’s Been

• Structural proteins Collagen

• Transportation Haemoglobin

Proteins Are Us

• In cells, when something needs to be done, it is a protein that does it.

• The human body contains over 30,000 different types of protein

• Other organisms have many of the same proteins as well as different ones

• Enzymes are the biggest class 3,000 enzymes in average mammalian cell ß-galactosidase is an enzyme

Transcription Factors Control expression of

genes Hormones

Control body function Antibodies

Fight infection Enzymes

Speed up chemical reactions

Carrier molecules Haemoglobin -carries

oxygen in the blood

Classes of Proteins• Structural

Collagen Found in bone and skin

Keratin ■ Makes hair and nails Fibrin

Helps clot blood Elastin

Major part of ligaments

Proteins AreDiverse In Structure

• Proteins can do many things because they are structurally diverse

• Differ in many properties: Size Shape Charge distribution Hydrophobicity Solubility properties

Variability ComesFrom Amino Acids

• Are polymers composed of 20 different amino acid building blocks

• As letters can be arranged in many ways, so too can amino acids

• Number, type and arrangement of amino acids determines structure and function

• Insulin has about 50 AA Most are >> bigger - from 100s to 1000s Allows for great diversity

Amino Acids

• All amino acids have a carboxyl group and an amino group

• A different R group is attached to each amino acid

Amino Acid

• R groups make each amino acid different• Some are:

Polar Nonpolar Charged Acids Bases

Twenty Amino acids

Folding

• DNA always has same structure• But proteins fold into many different shapes• Folding depends ultimately on amino acid

composition• Structure of proteins determines function• Structure allows proteins to

BIND to other molecules RECOGNIZE other molecules

Protein StructureAnd Function

Shape Is Critical

• Change in one amino acid can change the structure of the protein with a large effect on function

Sickle cell anemia

Image fromMedline Plus

Sickle Cell Anemia

• Single DNA base pair is mutated• Therefore one amino acid is altered • Glutamic acid is switched to valine• Glutamic acid is negatively charged, valine is

neutral• Changes how hemoglobin packs in cells• Alters shape of red blood cells when oxygen is

low.

Levels of ProteinStructure

• Protein structure is complex and important, so it is classified into:

1° - Primary 2°- Secondary 3° -Tertiary 4°- Quaternary

Primary Structure

• Linear sequence of amino acids• Peptide bond (covalent bond) holds it

together• Beads on a string

Primary Structure

Peptide Bonds R O H R l ll l lNH2 –C – C – 0 –H + H –N – C—COOH l l H H

R O H R l ll l l

NH2 -- C –C – N – C – COOH + H2O l l H H

Secondary Structure

• Regular repeating patterns of twists or kinks of the amino acid chain

• Examples Alpha helix Beta pleated sheet

Secondary Structure

• Hydrogen bonds hold structure together Weak, noncovalent, molecular interactions

• Hydrogen atom is bonded to an electronegative atom (like F, O, N) that is also partially bonded to another atom (usually also F, O, N)

Figure from National Human Genome Research Institute, by artist Darryl Leja. Used with permission. 

TertiaryStructure 3°

• 3-D Globular Configuration formed by bending and twisting of the polypeptide chain

• Stabilized by: Hydrogen bonds Electrostatic interactions

1. (Positive and negative) Hydrophobic interactions Sometimes covalent bonds

1. Disulfide bonds

QuaternaryStructure 4°

• Two or more polypeptide chains associate with each other

ß-Galactosidase

• Link to Protein Data Bank for picture of molecular image of ß-galactosidase

• www.pdb.org

Higher OrderStructure

• Higher order (secondary, tertiary, quaternary) structure is relatively “weak”

• In nature, “weakness” of noncovalent interactions is important

Flexibility1. Enzymes change shape when binding to

their substrates2. Necessary for proper function

How Proteins Lose Normal Structure And Function

• Primary structure hard to disrupt; covalent bonds are strong

How Proteins Lose Normal Structure And Function

• Can be broken apart by enzymes (proteases) that digest the covalent peptide bonds

Called proteolysis Occurs naturally in digestion Can be a problem in the lab; proteases can

destroy protein of interest Use cold to avoid proteolysis

How Proteins Lose Their Structure And Function

• Sulfur groups on cysteines may undergo oxidation to form disulfide bonds that are not normally present

• Proteins can aggregate leading to precipitation• Proteins can adsorb (stick to) surfaces

Higher OrderStructure In Lab

• Loss of higher order structure is denaturation• Denaturation occurs fairly easily

Affected by changes in pH Ionic strength Temperature

• May or may not be reversible

Denaturation

ManipulatingHigher Order Structure

• Often manipulated in lab Destroy folding when we do PAGE Use buffers to maintain the structure Use cold temperatures Add reducing agents to prevent unwanted

disulfide bonds in the lab --DTT or -ME

Analyzing Protein Structure

• X-Ray Crystallography Like a CAT scan in medicine X-ray taken at multiple angles and

computer uses the data to calculate a 3D image

• Nuclear Magnetic Resonance Like an MRI in medicine

X-rayCrystallography

• Isolate and purify protein• Form a crystal of the protein

Molecules of the protein are arranged in an orderly lattice1. Dissolve protein in solvent2. Precipitate into a crystal

• X-Ray the crystal

X-rayCrystallography

• Analyze diffraction pattern using software Make electron density map

• Process used to take years Different versions of crystal for comparison

1. Each with different heavy metal in lattice to provide reference point

• New X-ray sources - synchrotrons have reduced data collection time to few days

X-rayCrystallography

• Synchotron - Argonne National Lab• Still takes weeks to go from gene sequence to

3-D structure• (mrsec website, nanotechnology at UW-

Madison)

StructuralGenomics

• Goal to solve thousands of structures a year• Large scale automation required• Syrrx - structural genomics company

Robot places a drop of protein into 480 wells

11,000 crystallization experiments in 24 hours

New robot - 130,000 a day

Nuclear MagneticResonance

• Similar to MRI - Magnetic Resonance imaging in medicine

• No need for crystals, proteins in solution• Works for relatively simple proteins