Unit 1 - Protein Synthesis, Structure and Function

Clinical connections

Effects of misfolding, ie disease

Specificity and Flexibility of proteins causes

Risks

Functional Protein

Contributes to cell outcomes in usual conditions with tolerable fail rate

Shape and Structure specificies

Function of the protein

N Terminus

Amino End

C Terminus

Carboxyl end, new ones add to this end

Amino Acids

R groups + H + Amino Groups + COOH

Hydrophobic Amino Acids tend to be in

The core of soluable proteins

Aromatic Amino Acids

• Phenylalanine

• Tyrosine

• Trytophen

Aliphatic Amino Acids

Hydrocarbon chains

• Valine

• Alanine

• Leucine

• Isoleucine

• Methionine

Basic Amino Acids are

Positively charged

• Lysine

• Arginine

Acidic Amino Acids are

Negatively charged

• Aspartic

• Glutamic Acid

Hydrophilic Aminos

• Asparagine

• Theonine (neutral at 7pH)

• Glutamine (polar amine)

• Serine

Cysteine

Disulphide bridges with other cysteine

Glycine

Very small to sqeeze into small spaces and proteins to bend

Proline

R group covalently bonds with amino group creating kink for structure

Histidine

Has amino diethyl that changes positive or negative depending on pH

Peptide bonds forms by

Condensation reaction between amino and COOH

Translation

1. Ribosome subunits assemble to read mRNA

2. tRNA enters A site

3. tRNA shifts to P site, amino chain shifts

4. Used tRNA shifts to E site to be ejected

Primary Structure

1. DNA to mRNA

2. Introns removed from mRNA

3. Exits to cytosol

Random Coil Structure

Periodically ordered structure of protein

Statistical Coil

Protein spends most of its time in a certain structure

Native Structure

Functional protein structure

Hydrophobic Effect

Clumping of nonpolar, noncovalent molecules to aqueous solution to decrease interactions with water that add up for strong stability of folded structure

Setae Fibres in a Geckos Foot

Induces LD dipoles to let geckos walk sideways

Secondarry Structure

Periodic folding of polypeptide into distinct, conserved, geo arrangements

Motifs

Combinations of 2nd structure

Alpha Helixes

Spiral, rod like structure by COO bonds with H 4 positions away, 3.6 aminos per churn

Beta Sheets

Planar structure with 2+ strands aligned by H bonds

Beta Pleated Sheets

Laterally packed beta stranger from H bonds between COOH and amino from backbone in adjacent beta strand

Intramolecular H Bonds

Beta sheet in the polypeptide

Intermolecular H Bonds

Beta sheet betwen polypeptides

Turns/Loops

Connectors of beta and alpha

Beta Turn

3-4 aminos connecting beta strands of sheet

Coiled-Coil Motif

2 alpha helixes wrap aorund each other because both r groups are amphipathic and hydrophobic face inwards when aminos are at position 1 and 4 in repeat of 7 aminos

Zinc Finger Motif

Alpha helixes and 2 beta strands form 2 positioned residues with zinc atom

Beta Barrel Motif

Barrel form when last beta strands forms H-bonds with first strand

Helix Loop Helix Motif

2 alpha helixes joined by a loop region by non covalent interactions between aminos and calcium ion

Tertiary Structure

3D arrangement of all aminos

Domain

Functional unit of protein associated with a unique function and fold independently

Functional Domain

Region with specific activity of protein

Structural Domain

Region with a recognizable shape

Sre protein regulates

Cell cycle

Sre Protein Structure

• Small and large functional kinase domains

• SH2, SH3 structural domains

Quaternary Structure

Number and organization of subunits in a multiple protein complex

Multimeric Protein

Functional protein with multiple polypeptides

Dimer

2 polypeptides or subunits

Trimer

3 polypeptides

Homodimer

2 same polypeptides

Heterodimer

2 different polypeptides

Instrinsically Unstructured Proteins

Proteins that lack teritary structure when alone

Post translation changes

Individual aminos r groups

Lysine + Acetyl

Acetyl Lysine, to protect proteins from proteases

Methylated Histidine Residues

3 methyl histidine

Phosphorylation

PO4 from ATP to OH group of serine, tyrosine, threonine by kinases

Proline can be

Hydroxylated, 3 of them create functional collagen

Carboxylation adds

Negative charge

Glycosylation

Add carbohydrates, protect proteins and helps folding

Sugar add to

OH groups of serine and threonine

Lipidation

Anchors proteins to hydrophobic biomembranes

Protein folding is

Spontaneous, reversible, and unique

Reversible Denaturation Experiment

1. Denatured protein with urea to break H bonds, and beta mercaptoethanol to break disulfide bridges

2. Dialysis to remove denaturants which brought it back to original shape

Villin

36 residue, alpha helical protein with hydrophobis core of 3 phenylalanines

Sickle Cell Anemia

Misfolded haemoglobin tetramer from glutamate to valine at position 6 causes it hydrophobic instead charged and forms polymers which gets stuck in capillaries

Challenges with Protein Folding

• Folding driven by chemistry

• Protein collisions

• Folding must match rate of translation

• Exposure of hydrophobic areas can cause other hydrophobic areas to stick

Cystic Fibrosis

Genetic disease from deletion of phenylalanine 58 of CFTR gene making it unable to bring Cl into cell causing thick mucus

Specificity

Ability to preferentially bind to few molecule only through shape, charge, flexibility

Affinity

Strength of binding, better fit = stronger, longer signal through surface matching and H bonds

Molecular Complementarity

Molecular shape fit well that favorable non covalent interactions form, both specificity and affinity dependent

Chaperons

Stop inappropriate interactions between amino acids and increase efficiency of protein folding

Molecular Chaperons

Monomeric proteins, binds to hydrophobic residues to stop wrong hydrophobic interactions

Heat Shock Proteins

Proteins for high temperature to help refold denatured ones

HSP 70

Nucleotide site for ATP, substrate site for folding using hydrophobic residues in HSP help hydrophobic patches to bind with ATP causing conformational change

DNA J/HSP 40

Stimulates ATP hydrolysis in HSP 70

Where is BiP

ER

Where is Dnak

Bacteria

Chaperonins

Large macromolecular complex with many different proteins to form chamber or barrels to fold in isolation

TCiP

Eukaryotic cytosol

GroEL

Bacteria or chloroplast

HSP 60

Mitochrondria, 1 subunit of GroEL = apical + intermediate + equatorial domain

HSP 60 Bacterial Chaperonin

2 GroEL + GroES cap and a hollow chamber inside

Chaperonin Catalyzation

1. Bottom releases GroES cap and ADP, the top chamber binds to ATP and peptide

2. GroES cap binds to the top of GroEL

3. Change enlarges chamber to allow it to fold inside

4. ATP hydrolysis allows GroES to come off and protein diffuses out, if misfolded the protein enters the bottom chamber

Tight Confirmation

GroEL without cap

Relaxed Confirmation

GroEL with GroES, larger

Protein Degradation

1. Protein tagged with ubiquitin

2. Proteasome recognizes ubiquitin and protein cut in 7-8 residue peptides

Ubiquitin

Small protein, 76 residues

E1

Ubiquitin activating enzyme that picks up free ubiquitin

E2

Ubiquitin conjugating enzymes that facilitates ubiquitin to protein attach

E3

A ubiquitin ligase made up of a bunch of proteins that recognize specific targets to degrade and attach it

Ubiquitinylation

Addition of ubiquitin using E1,E2,E3

Ubiquitinylation Steps

1. E1 attaches to ubiquitin using ATP

2. Ubiquitin transfers to E2

3. E3 recognizes, captures protein and E2 and E3 interacts and ubiquitin transfers from E2 to protein

4. Repeat many times for the same protein

Proteasome

Central hollow cylinder with caps on ends, forming narrow openings which unfolded polypeptides are threaded through

Proteasome Steps

1. Protein recognized by cap enters the proteasome unfolded and ubiquitin removed as it goes in

2. Cleaves non specific proteolytic activity

3. Small peptides further degraded by cytosolic proteases or in lysosome

Spinocerebellar Ataxia

Mutation in ataxin 1 gene causes misfolded ataxin protein but can’t be unfolded by proteasomes causing build up and lethal aggregation

Ligand Binding Pocket

Area where ligand binds, protein with cAMP pocket has 6 chains h bonding to areas of cGMP

Binding Affinity

Free energy of interaction between a protein and ligand, measured by binding

Higher K_eq

More ligands bound to protein

K_d

Dissociating constant, strength of affinity

V_max

Maximal rate of reaction because all enzymes are saturated

Takes higher concentration for a

Low affinity enzyme to get to V_max as more dissociation