Chapters 4-6? Flashcards
Fucntions of Proteins
- Enzymes(-ase)
- Signaling
- Receptors
- Structural
- Transport
- Storage(fats stored in vessicles)
- Proteins make up a majority of stuff in bacterial cells(15%)
- the shape of a protein determines its function
- proteins are built from amino acids joined by peptide bonds
ATP Synthase
- uses mechanical energy to make ATP
- proteins span plasma membrane
- are in phospholipid bilayer so must have some hdrophobic residue
Proteasomes
- large protein “containers”(trash cans) where inside other proteins are degraded
Kinesin
- motor protein
- transports cellular cargo like organelles
Proteins can denature(unfold) and renature(refold)
- When a purified proteins isolated from cells is exposed to a high conc. of urea it denatures
- when we remove the urea it renatures
- proteins can also be denatured by change in pH/heat
- Noncovalent bonds break first
- there are specific conditions for refolding
- chaperones help proteins fold correctly
Misfolded proteins can cause disease
- if you consume misfolded proteins your proteins begin misfolding
- mad cow disease
- bovine spongiform encephalopathy
- infectious neurodegenerative disease
- Alzheimer’s Disease
- plaques in brain amyloid beta protein aggregation
Interactions stabilizing tertiary structure
- The final shape is determined by a variety of bonding interactions between the “side chains” on the amino acids
- Hydrogen bonds
- Ionic Bonds
- Disulphide Bridges
- Hydrophobic Interactions and Van der Waals
Interactions stabilizing secondary structure
- backbone of hydrogen bonds
Secondary structure alpha helix
- Hydrogen bonds backbone
- N-H to C=O, N+4 same chain
- 1 turn = .54nm; 3.6 residues
- side chains point out and towards N-terminus
- Handedness(right or left)
Secondary structure beta sheet
- Hydrogen bonds backbone
- N-H to C=O, adjacent chains
- 1 pleat=0.7nm(distance btwn two R-groups on 1 side
- Side chains alternate on either side of sheet
- Parallel or antiparaller
Amphipathic motifs
- coiled-coil
- EX: Keratin(wool, horns)
- transmembrane helical proteins
- opioid receptor(brain)
- binds opiates(morphine heroin)
- mediates responses
- ex: altering the perception of pain
Proteins are often composed of multiple domains
- protein domain: any segment of a polypeptide that can fold independently into a compact, stable structure
- have distinct functions
- 40-350 amino acids
Intrinsically disordered(unstructured) regions
- have many functions
- binding
- tethering domains within a protein
- tethering interacting proteins
protein assemblies
- Ebola and Zika
Protein machines
- chaperones, motor proteins, ribsomes
proteins show ligand binding specificity
- Binding site compatibility:
- size
- shape
- specific interactions
- optimal binding by ligand satisfies ALL available bonds
Enzymes bind to one of more ligands
- these are called substrates
- EX: lysozyme
- hydrolase
- natural antibiotic
- substrate: polysaccharide chains
- cleaves via hydrolysis
enzymes catalyze reactions in many ways
- enzymes catalyze rxns by:
- orients two substrate molecules
- rearrange charges in substrate
- changes shape of substrate
- lysozyme does so to stabilize transition state
related enzymes share similar properties
- enzyme family members
- catalyze similar rxns(do similar chemistry)
- EX: all kinases catalyze addition of phosphate groups
- have similar active/binding sites
- EX: many hydrolases(like lysozyme) have same amino acids in their active site
- catalyze similar rxns(do similar chemistry)
5*Drugs are often protein ligands
- ligand: any substance bound by a protein
- EX: small molecules, other proteins, etc.
- Drug = Ligand
- Rational drug design
- using knowledge of a bimolecular target(often the protein structure) to design a drug
- EX: Gleevec and nilotinib treat Chronic mylogenous leukemia(CML)
- CML is cause by chromosomal mutation
5*targeting an enzyme to stop cancer
- Qs to ask:
- what is the enzyme’s function?
- how does the enzyme complete its function?
- does the enzyme have a ligand?
- We must solve crystal structure of protein target with or without ligand
- Can exploit differences in targeting
- EX: an electrostatic interaction vs a hydrogen bond in one position
- can also exploit similarities
5*Targeting Abl: Kinases Bind and Hydrolyze ATP
- Novartis first used all available kinase structures
- designed library of potential kinase inhibitors
- High-throughput screening for inhibition of Bcr-Abl
- structurally characterized Abl kinase domain with hits
- Hits from screen(Pyrimidine A) were a precursor to the optimized hit of imatinib(Gleevec; Glivec)
5*Gleevec: the miracle cancer drug
- Before Gleevec: 5 year survival rate-30%
- With Gleevec-89%
- Was fastest approved drug by FDA
- BUT, discover some patients are immune to it’s effects! How?
- random mutations in the BCR-Abl protein
5*Nilotinib
- Second generation CML Drug
- developed using the structure of Gleevec bound to Abl kinase domain
Feedback inhibition for regulation of enzymes is essential
- BCR-Abl always “on”
- leads to cancer b/c of unregulated cell proliferation
- negative or positive
- leads to cancer b/c of unregulated cell proliferation
Enzymes can be regulated Allosterically
- Allosteric regulation: regulatory molecule binds to site other than enzyme active site, induces conformational change
- non-competitive inhibition
- can be negative or positive
Proteins can be regulated via covalent modifications
- hundreds!
- EX:
- phosphorylation
- acetylation
- ubiqutination
- methylation
DNA structure: the race
- Linus Pauling- Caltech-1951 proposes correct model for protein structure. Working on DNA.
- James Watson and Francis Crick-Working at Cavendish Labs on a model
- Maurice Wilkins-At King’s working on DNA, X-ray crystallographer
- Rosalind Franklin-biophysicist, X-ray crystallographer-comes to King’s College to work
Franklin’s X-ray photo 51 solved structure of DNA
- Tension btwn Franklin and Wilkins
- Anti-female culture at King’s college
- EX: Franklin not allowed to eat in some areas
- Diffraction pattern, shown without her knowledge to Watson and Crick
- Leaves DNA work, collaborates with Arthur Krug on viruses
- Succumbed to ovarian cancer in 1958
Watson’s Book: “The Double Helix”
- Published after Nobel
- Initially rejected by publishers
- Wilkins, Crick also objects
- Portrayal of Rosalind Franklin
- asserts Franklin was not smart enough to interpret her own data
- her notes indicate she had correctly interpreted data
DNA replication is semiconservative
- occurs in nucleus, during interphase of cell cycle
DNA synthesis begins at replication origins
- Replication origins are:
- 100 bp: simple organisms
- variable length in humans
- A-T rich
- Bacteria: 1 replication origin
- Humans:~10,000
DNA Helicases and Single-stranded Binding proteins at origins
- DNA helicase uses ATP hydrolysis to unwind helix
- Single-stranded binding proteins(SSB) bind to unwound region
- Essential to replication initiation
Consequences for DNA replication process if each of the following were missing:
1) DNA polymerase
2) DNA ligase
3) Sliding clamp
4) Nuclease
5) DNA helicase
1) DNA polymerase: interrupt DNA synthesis
2) DNA ligase: Okazaki fragments would not bind back together
3) Sliding clamp: might begin replication, but DNA polymerase will come off so you will have short DNA breaks(see more in lagging than leading b/c lagging uses sliding clamp more often)
4) Nuclease: affects removal of RNA primer and integration of new nucleotides, wouldn’t have a repair process
5) DNA helicase: couldn’t unwind double helix and process wouldn’t start
DNA polymerase adds nucleotides to the 3’ end
- DNA polymerase helper proteins:
- sliding clamp keeps DNA polymerase on strand
- clamp ladder helps sliding clamp assemble
- has proofreading activity
Replication forks are assymmetrical: leading and lagging strand
- Leading strand
- template is 3’-5’
- continuously synthesized
- Lagging strand
- template is 5’-3’
- synthesized in small Okazaki fragments
RNA primers are needed for DNA synthesis
- Primase:
- RNA polymerase
- short complementary RNA primers
- needed to initiate replication
- needed for Okazaki fragments
Telomeres and Telomerase
- Telomeres shorten over time
- every cell division it shortens by ~30-200bp
- “Molecular clock”
- max cell divisions per cell: 50-70 times
- Telomerase is not made in most cells
- exception: egg and sperm cells
- Cancer cells switch on telomerase production
Spontaneous DNA damage
- Spontaneous: naturally occurring mutations.
- EX: depurination
- Loss of entire purine base(A or G)
- Sugar-Pu bond less stable than sugar-Py
- result: may stall replication
- Loss of entire purine base(A or G)
- deamination(loss of amine,NH3)
- removal of amino group/amine from base
- deamination of cytosine
- removal of amino group/amine from base
- EX: depurination
Induced DNA damage
- Induced: exposure to physical or chemical agent(mutagens) that interact with DNA to cause a mutation
- EX: radiation, chemical mutagens
- EX: UV radiation
- causes pyrimidine dimers
- Result: DNA polymerase stalls
Xeroderma Pigmentosum
- Inability to fix UV damage
- Recessive genetic disorder where DNA repair enzyme mutated
- Cannot repair normal DNA UV damage (thymine dimers)
- Multiple skin cancers
- “Children of the Night”
- can’t be exposed to UV rays from sun
DNA repair
- Mismatch Repair
- repairs single-strand damage
- nuclease helps remove incorrect nucleotide
- DNA (repair) polymerase fills in space
- DNA ligase seals space
- repairs single-strand damage
protein folding
- hydrophobic forces:
- polar aa side chains tend to be displayed on the outside of the folded protein, so they can interact with water
- nonpolar aa side chains are buried on the inside to form a highly packed hydrophobic core of atoms that are hidden from water
- weak noncovalent bonds: H bonds, electrostatic attractions, Van der Waals attractions
- rly weak, so a large number of them is required
- H bonds can form btwn water and polar side chains on outside
- final folded structure = conformation
- goal: minimized G(free energy)
