BCH210 Midterm

Function of proteins

Function of proteins

Determined by their structure.

Enzymes do catalysis

Hormones transmit signals.

Structural proteins give cells their structure.

Types of protein bonds/interactions

1. Covalent: Hold the amino acids together and are strong.

   Ex. Peptide bonds

2. Non-covalent: Allow the polypeptide chains to fold into the protein.

   • Ionic bonds: Form between positively and negatively charged amino acids. Allow ligands, cofactors, and substrates to bind. (Salt bridges)

   • Hydrogen bonds: Form between H and N, O, or S. (Between side chains)

   • Hydrophobic: pushes hydrophobic a.a to the inside

Cofactors

Molecules in proteins that help with structure and function.

1. Coenzyme: Shuttle functional groups to help with protein function.

   • Prosthetic Groups: Large molecules that are bound tightly to the protein

   • Cosubstrates are bound loosely.

2. Metal ions: interact with the protein to either help with structure or catalysis (function).

   • Mg, Ca, Co, Zn.

Importance of Water

• Polar → can form hydrogen bonds → can be a hydrogen bond donor or acceptor

• Can solubilize other molecules that have hydrophillic functional groups.

• Is polar → avoids hydrophobic molecules → makes proteins fold.

Hydrophobic Effect

Interactions between hydrophobic functional groups that exclude water.

Allows the formation of macromolecular structures.

Obeys the 2nd law of thermodynamics since it increases the entropy of water.

Acid-Base reactions

H+ and OH- ions are formed.

pH

pH = -log [H+]

Is a measure of aciditiy

Low pH → more acidic → more H+ ions that can protonate functional groups.

pKa

pKa = -logKa, Ka = [H+][A-]/[HA]

Measure of acid strength.

Strong acid → High Ka → Low pKa.

Henderson-Hasselbach Equation

Calculates pH in relation to pKa.

pH = pKa + log [A-]/[HA]

Protonation/Deprotonation of Functional groups

Functional groups in proteins need to be protonated or deprotonated for interactions to occur.

pH < pKa → environment is highly acidic → lots of H+ that can protonate functional groups → protonated.

pH = pKa → no change

pH > pKa → environment more basic → low H+ and high OH → functional group is deprotonated.

Buffers

Buffers are a combination of weak acids and conjugate bases that help maintain the structure of a protein.

Neutralize small additions or losses of H+ → pH stays the same → functional groups aren’t protonated or deprotonated → structure remains unchanged and function doesn’t change.

Bohr Effect

Change in Hemoglobin’s ability to carry oxygen.

Low pH → His146 is protonated → forms a salt bridge with Asp94 → structure changes → oxygen released.

However, CO2 that is found in the tissue can combine with water to make H+, lowering the pH and bringing back hemoglobin’s function.

Amino acid composition

Made of an amino group, carboxyl group, and an R group.

Essential: comes from diet.

non-essential: can be made from other amino acids.

Glycine

Gly, G

Hydrophobic/Non-polar

Alanine

Ala, A

Hydrophobic/Non-polar

Valine

Val, V

Hydrophobic/Non-polar

Leucine

Leu, L

Hydrophobic/Non-polar

Isoleucine

Ile, I

Hydrophobic/Non-polar

Methionine

Met, M

Hydrophobic/Non-polar

Proline

Pro, P

Hydrophobic/Non-polar

Phenylalanine

Phe, F

Hydrophobic/Non-polar

Tryptophan

Trp, W

Hydrophobic/Non-polar

Serine

Ser, S

Hydrophilic/Polar

Threonine

Thr, T

Hydrophilic/Polar

Tyrosine

Tyr, Y

Hydrophilic/Polar

Cysteine

Cys, C

Hydrophilic/Polar

Asparagine

Asn, N

Hydrophilic/Polar

Glutamine

Gln, Q

Hydrophilic/Polar

Histidine

His, H

Positive

Lysine

Lys, K

Positive

Arginine

Arg, R

Positive

Aspartate

Asp, D

Negative

Glutamate

Glu, E

Negative

Disulfide Bonds

Form between sulfhydryls of free cysteines.

Bonds formed in an oxidation (LEO) and broken in a reduction (GER).

Product is called a cystine.

Stabilizes structure.

Hydrophobic vs Hydrophilic amino acid locations

Hydrophobic amino acids are found on the interior while hydrophilic amino acids are found in the exterior.

Hydrophobic can be on the exterior sometimes to help with non-covalent interactions.

Post translational modifications

Affect the function of a protein.

1. Phosphorylation

2. Ubiquitination: Adds ubiquitin which is a marker for destruction.

3. Glycosylation: adds a sugar.

Mutations

1. Silent: Changes the 3 letter code but creates the same amino acid.

2. Non-conservative: changes the 3 letter code and the amino acid.

3. Conservative: changes the 3 letter code and amino acid but maintains function.

Zwitterion

Molecule that has charged components but an overall neutral charge.

Buffer Regions

Same as the number of functional groups.

Each pKa value is a buffer region

Isoelectric point

the pH at which the molecule is neutral

pI = (pK2+pK3)/2

Primary structure

Linear chain of amino acids held together by peptide bonds (polypeptide chain)

The polypeptide chain starts at the n-terminus.

Amino acids are joined together in a condensation reaction, where the n-terminus and c-terminus of 2 different amino acids form a bond.

The peptide bond that holds the amino acids together is polar and uncharged. It acts like a double bond due to its resonance. It is rigid and planar, meaning it can’t rotate.

The psi bonds in the carbonyl and the theta bond in the amine, however, can rotate. If they rotate to form the trans conformation, steric strain is minimized.

Secondary Structure

Hydrogen bonds

Alpha helices, beta sheets, and beta turns.

Alpha helices are coils of proteins and the hydrogen bonds occur in the same chain

• The carboxyl group of one residue forms a hydrogen bond with the amine group 4 residues away. (i and i+4)

• Proline usually isn’t in alpha helices because it is really rigid and can’t rotate. Also, there is no hydrogen to form hydrogen bonds.

• Alpha helices can be polar, hydrophobic, or amphipathic depending on the amino acids present.

Beta sheets are sheets of proteins and the hydrogen bonds occur between chains.

• hydrogen bonds link the carboxyls and amines of nearby chains.

• can be parallel of the n-termini are aligned and antiparallel if the n-terminus and c-terminus are aligned.

Beta turns are a 4 residue segment that alows peptide bods to actually rotate.

• The rotation causes hydrogen bonds to form between carboxyl and amine.

Tertiary structure

Disulfide bonds, electrostatic, hydrogen bonds, hydrophobic

Combination of secondary structures through non-covalent interactions.

Ionic interactions occur between positively and negatively charged amino acids.

Hydrophobic interactions occur between non-polar amino acids to put them in the center.

Hydrogen bonds occur between the n and c terminus of amino acids.

Ligands can also be used to bring the secondary structures together.

Disulfide bonds stabilize the structure

Protein domains

Commonly seen structures.

Proteins with the same domains have the same function.

Protein stability

Ligands can be used to change the structure of proteins to make it more energetically favourable (like sitting instead of squatting)

PTMs can be used to modify the a.a. sequence by adding things.

Different types protein structures

Globular proteins: Compact and spherical

• Act as enzymes, hormones, and antibodies.

Fibrous proteins: Have an elongated and fibrous shape.

• have a structural role.

Transmembrane proteins: within the membrane

Hemoglobin

Is an example of a non-polar globular protein.

Takes oxygen from the lungs to the blood.

The heme group binds covalently to the hemoglobin and is responsible for picking up the oxygen

Oxygen binds to heme → the iron in heme shifts → pulls histidine down → makes it easier for new oxygen to bind.

Antibodies

Made of 4 chains; 2 light chains and 2 heavy chains. Interconnected by disulfide bonds

Form a Y shape.

The tips of the Y are the antigen-binding site.

Each antibody has a randomized binding site sequence, allowing different antibodies to recognize different antigens.

G protein-coupled receptors

Used to recognize the binding of ligands like adrenaline.

• In the extracellular space, there is a ligand binding domain that changes conformation when a ligand binds → conformational change activates the g protein.

Needed because ligands are polar and can’t move through the non-polar membrane.

Why purify proteins

There are bags of proteins in the cell with lots of membranes and organelles that can interfere → by purifying proteins, we can isolate the protein and prevent any interference.

Cell type for purification

Must choose the right cell type or tissue for getting the right amount and quality of protein.

Use the cell type that is specific to the protein of interest.

Cell lysis

Breaking open the cell.

1. Grinding: Rips open

2. Sonication: Frequency to burt

3. Vortexing with glass beads: Spins very fast to rip open

But these make a lot of heat which can cause the protein to lose its function.

4. Osmotic pressure: Difference in solute concentration inside and outside the cell → increases pressure → cell bursts

But cell also loses function.

5. Chemical detergents.

All cell lysis methods are hamrful because when the cell bursts open, theres lots of free proteins interacting.

• May release proteases. But protease inhibitors can prevent degradation

Protein Absorbance

Proteins can’t absorb visible light but they can absorb UV light.

Beer lambert’s law can be used to find the concentration of a protein based on the absorbance of light by their aromatic amino acids.

Beer lambert law

A=Ɛ c l

Ɛ = light absorbed

c = concentration

l = pathlength of cuvette

Size Exclusion Chromatography

Seperates proteins based on their size.

Coloumns of beads that trap the proteins

Smaller proteins enter the beads and get stuck there, making them move down slower.

Large proteins are too big to enter the beads so they don’t get stuck and move down quickly.

Ion-Exchange chromatography

Seperates proteins based on their net charge.

Cations (-) are used for positive proteins while anions (+) are used for negative proteins.

The cation or anion beads trap proteins of oppsoite charge → anything with a different charge flows out → pH changes to release the proteins that were stuck.

Affinity Chromatography

Separates based on binding interactions.

Resins have a ligand bound to them → the protein of interest forms a non-covalent bond with the ligand → traps only the protein of interest → protein of interest then released by adding free molecules that outcompete the protein for binding.

Used for proteins in small volumes.

SDS page

Helps visualize proteins.

SDS is a detergent that denatures proteins.

Makes everything negative so charge can be ignored and size can be the focus.

BME added to break the disulfide bonds.

Immuno-blotting/Western Blotting

SDS gel used to make the polypeptides negative → separate based on size → transferred to a polymer sheet → creates mirror image → primary antibody added → secondary antibody added to help visualize the proteins → illuminated and fluorescence is measured.

To prevent the proteins from sticking to the antibodies, they are blocked with milk.

Mass Spectrometry

shoot peptides with lasers of high energy electrons → measure their time of flight.

Time of flight is dependent on size and charge

Smaller = faster.

IR spectroscopy

Shows the secondary structure of a protein

IR light has specific wavelength → shoot the protein with the light → makes the peptide bonds shake → shaking of protein emits its own frequency.

Alpha helix: 1650-1660

Beta sheet: 1620 - 1640

Antiparallel beta sheet: 1675-1695

Random coil: 1640-1650.

Circular Dichroism

used to see the secondary structure of a protein.

Light can be circular if it turns left and right.

Light passes through the protein → absorbed at a specific wavelength in each structure.

Alpha helix: 222 and 208 for NEGATIVE and 195 for POSITIVE

Beta sheet: 217 negative

Random coil: 198 negative

Xray crystallography

Used to find the tertiary or quaternary structure of a protein.

In certain conditions with specific pH and temperature, proteins can form crystals.

Crystals form → placed in a machine → produces patterns that show coordinates of structures → angles determine what the structure is.

NMR

Used to find the tertiary and quaternary structure of a protein.

Proteins absorb electromagnetic radiation → shows the environment of the nucles.

Can only be used of small proteins < 25 kDa.

Cryo-electron microscopy

Used to find the tertiary or quaternary structure of a protein.

Thin layer of purified protein is placed on a fine grid → frozen to trap molecules in their orientation → beam of electrons passed through.

Used on larger complexes > 100 kDa.

Chromophores

Functional groups that can absorb UV light.

Aromatics, amide carbonyls.

The indole ring in tryptophan.

Red shifted in polar environments.

Florescence microscopy

Shows proteins in their natural environment.

Proteins are tagged with fluorescence to show their location but not their interactions.

Antibodies with fluroescence can also be used as tags.

FRET and BRET

show protein interactions.

FRET uses emission of light from a fluorophore to excite an acceptor

BRET uses enzymes.