Glossary of Part 1b
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- Glucose - two forms
- B(beta)-form = CELLULOSE
a(alpha)-form = starch
Beta means up position; axial
Alpha means down posit; equatorial
- Nucleic acids
- Backbone: sugar-phosphate-sugar-phosph..
Differentiation: comes from bases
- Small, water-insoluble molecules
-Phospholipids form extended bilayers
-Structures include long chain FATTY ACIDS
- Years ago Earth was formed
- 4.5 Billion
- Years ago Microorganisms formed
- 3.5 billion
- Eukaryotes formed
- 2.25 billion yrs ago
- Oxygen atmosphere formed
- 1.5-2 billion yrs ago
- Early steps in Evolution
- -Prebiotic Synthesis of Molecules
-Biosynthetic Cycles began
---CELLULAR LIFE (the RNA world?)
- Urey-Miller Experiment
- Illustrated that simple, prebiotic conditions can generate amino acid products.
-Reducing Atmsphere of NH3, H2, CH3, H2O
-Generated Amino Acid products
- Hammerhead Ribozyme
- -Discovery suggests that catalytic RNA molecules could have played fundamental roles in evolution of life.
-Makes plausible the idea of an early "RNA WORLD"; lifeforms depended on RNA for heredity, info storage, promotion of specific reactions.
- Imino acid
-3 carbon cyclic side chain
-only amino acid with side chain bonded to amino group.
- 2 Ways to make a Buffer
- Mix an Acid with its salt to get desired pH.
Mix weak acid or base w/ strong base or acid.
- Nonpolar amino acids
- Glycine, Alanine, valine, leucine, isoleucine, methionine, proline
- only non-chiral amino acid
- glycine; not L or D
- aromatic amino acids
- phenylalanine, tyrosine, tryptophan
- Special features of Tyrosine/Tryptophan
- -kind've polar - Hydroxyl/NH groups.
-Aromatic rings contain delocalized electrons that strongly absorb UV lite.
Allows determination of protein concentration in solution. (Beer's law)
- Polar Uncharged Amino acids
- Serine, Threonine, Cysteine
- Cysteine special features
- Side chains S-H oxidize to form disulfide bonds/bridges.
- Loss of electrons (H+)
- Polar Amino Acids with Basic R chains
- Lysine, Arginine, Histidine
- Polar Amino Acids with Acidic R chains
- Glutamate, Aspartate
- Nonpolar Amino Acids additional
- Asparagine, Glutamine
- Water is a Weak Acid
- just know that
- % of Other ions, metabolites in
-Total cell composition
-Dry mass composition
- Major classes of biomolecules
- -Linear polymers of 20 different amino acids
-Chemically, structurally, functionally diverse
- -Very small
-Highly structured intermolecular Hydrogen bonding
- -Linear or branched chains of SMALL SUGARS
-Gives cells STRUCTURE
- ATP SYNTHASE function
- -A molecular machine
-Functions as a ROTARY ENGINE, synthesizing ATP as it spins
- ATP Synthase Structure
- 8 DIFFERENT POLYPEPTIDE CHAINS
22-24 Total Subunits
-1 each: a, y(gamma) delta(d) E(epsl)
-3: alpha, beta
-10-12 copies of C
- Amino Acids
- Building Blocks of Proteins.
Residues hooked together to form long polypeptide chains.
- Dominant Amino acid conformation
- L-alpha amino acid
other is D
- Polypeptide Backbone
- Number of E.Coli bp
- 4.5 x 10e6
- Number of Human base pairs
- 3 x 10e9
- % water in cell composition (total)
- % water in dry mass cell composition
- 0 obviously, it's dry
- % of Protein in
-Total cell composition
-Dry mass cell composition
- Total = 20%
Dry = 66%
- % of Lipids in
-Total cell composition
-Dry mass cell composition
16% in dry mass
- % of Carbohydrates in
-total cell composition
-dry mass cell composition
- % of Nucleic Acids in
-Total Cell composition
-Dry mass cell composition
- + charged ions migrate to...
- charged ions migrate to...
- Anode - this occurs below the pI
Cathode - occurs above the pI
- Native Protein Structure
- the particular folded structure of a protein under biological conditions
- 2 Examples of Fibrous Proteins
- Collagin and Keratin
- the acid dissociation constant of a SIDE chain on an amino acid - the r group
- Structural relative of Hemoglobin
-Stores oxygen in muscles
-predominantly alpha helices
-1 single chain.
- alpha Keratin
- -in hair and nails
-has long coiled helices, fibrous protein, no compact folding.
-an alpha-helical coiled coil - alpha helices intertwined to form long fiber
- 4 levels of protein structure
- Primary Protein Structure
- sequence of amino acids
- Secondary Protein Structure
- regular, local folding of peptide backbone.
- tertiary protein structure
- compact folding of a single pp chain.
-aka, domain structure.
-Pp chain can have multiple domains.
- Quaternary Protein Structure
- multiple individually folded chains (subunits) are tightly associated.
- Immunoglobulin domain
- Only Beta-barrels. No helices.
Formed of 8 pp strands.
- Beta clamp
- A subunit of DNA polymerase
- 4 Non-covalent Intermolecular Forces that stabilize Protein Structure:
- Hydrogen bonds
Van der Waals forces
- Hydrogen bond
- -bond that forms between a polarized h-bond donor and H-bond acceptor with unshared electrons.
-Length: Donor-hydrogen: 0.9A
Geometry: 180degrees SP
- H-bond length
- 2.0 A
-That's between the H and the acceptor.
-Overall = 2.9
-From H-donor is 0.9
- Ionic Bonds/Salt bridges
- strong interaction between two oppositely charged ionic groups.
-Energy depends on distance btwn molecules, dielectric constant of the solvent.
- Van der Waals forces
- weak nonspecific interactions from transient charge fluctuations in electron shells.
-criticaly depends on distance between atoms/groups of atoms.
- Contact distance
- how close 2 atoms can get to each other before their van der waals forces become repulsive.
- Hydrophobic interactions
- apparent interactions between nonpolar molecules. But not really.
-Really the highly structured H-bonding of water.
- Oil-drop effect
- To maximize energy of a system of protein and water: water-molecular surface interface is minimized by nonpolar areas aggregating. Reduces entropy, maximizes H2O's freedom to interact.
- Linus Pauling
- -discovered that peptide bond C-N is PLANAR and ALWAYS TRANS.
-has a PARTIAL DOUBLE BOND due to resonance of the pi electrons of the carboxyl group.
- A.A. that is exception to the Always Trans, Always Planar Peptide Bond rule:
- PROLINE Energy difference is only small between trans/cis; cis has a little more steric hindrance than trans.
- He predicted 3 secondary structures of pp backbones that maximized H-bonding and had correct geometry.
- Who is Linus Pauling, 1990-1994?
That is correct.
..Paved the way for double helix..
- 3 2ndary structures
- Alpha helix
Parallel Beta sheet
Antiparallel beta sheet
-All result from Hydrogen bonds
- Alpha Helix Features
- -Each A.A. is H-bonded to 4th residue in sequence.
-3.6 residues per turn
-5.4 Angstrom Pitch
-R-groups (sidechains) stick out away from axis.
- B(A) and B(P) sheets
- B(A)=Antiparallel; 2 pp chains running opposite; H-bonds are straight between corresponding residues; every other.
B(P)=Parallel; much longer than anti;
-Both have repeat every 2 residues
- Phi angle
- between Alpha Carbon and Nitrogen
between Alpha Carbon and Carbonyl C
- Which is more common:
Right or Left-handed helices?
- RIGHT. Left is very rare.
- Torsional angles determine..
- Backbone conformation.
Phi and Psi
- Structural relative of Myoglobin
-transports oxygen in blood.
-Tetramer - 2 alpha, 2 beta.(4 subunits)
Subunits are structurally similar to myoglobin's 1 unit. (predominantly alpha helices)
- X-ray crystallography
- 1. Grow crystals
2. Collect data - send X-ray through crystals, diffract, beams go around different e- densities and film picks up remaining rays.
3. Use Diffraction Pattern
4. Get E- density map, fit molecular structure.
- What directs tertiary folding?
(into globular proteins)
- Hydrophobic intercations.
- Oildrop Model of Globular Proteins
- Tertiary Folding; Directed by Hydrophobic interactions. Nonpolar molecules pack densely into protein's interior, polar on exterior. Minimizes surface area btwn water and nonpolar.
- Bonding that aids Tertiary folding
Ionic - strong when protected from water (otherwise it hydrates them).
VanderWaals - tight packing interior of protein; atoms rub shoulders.
- membrane proteins that have reversed distribution of polar and nonpolar groups.
-Nonpolar is EXTERIOR - hydrophobic (in contact w/ membrane)
-Polar is INTERIOR - hydrophilic
-creates H2O channel inside protein.
- Not only do Myoglobin/Hemoglobin have similar tertiary folding.. also have..
- Similar amino acid sequences.. hmm what does that tell us?
-Specific amino acids can predict secondary structure AND tertiary structure.
- What types of secondary structures do Glycine/Proline contribute to?
- SHARP TURNS.
-They're alpha helix breakers.
- -Demonstrated spontaneous folding of native protein globular 3ary structure.
-Used ribonuclease A, Urea, B-mercaptoethanol.
- Ribonuclease A
- A digestive enzyme, synthesized in the pancreas.
-8 Cysteins, with 4 Disulfide bonds.
-Predominantly a B-sheet structure.
- Protein Denaturation Reagents
(in anfinsen experiment)
- -Excess B-Mercaptoethanol.
- -reduces disulfide bonds in proteins.
-used by anfinsen on ribonuclease A
- Disrupts H-bonding and hydrophobic interactions.
-Great h-bond former itself.
-Interrupts Lattice structure of H2O - so it disrupts the hydrophobic effect.
- -removes urea from denatured proteins.
-Small molecules (MW<6000) diffuse out of porous tubing. The desired protein stays inside the tubing.
- transmissable spongiform encephalopathies
- Mad Cow (BSE)
Kuru (human, New Guinea)
variant Cruetzfeld-Jakob Disease (human)
-Associated with PRIONS
- -Infectious agents
-Contain no detectable DNA or RNA
-Misfolded cellular proteins in alternate conformation
- PrPc and PrPsc
- two prion structures.
PrPc = normal; 3 alpha helices
PrPsc = pathogenic; 2 alpha helices, one from normal switches to Bparallel sheet that's very stable.
- Prion Hypothesis
- -Association of PrPsc the pathogenic form with PrPc the normal form converts the normal to pathogenic too.
-Result: neurotoxic filaments grow.
-Causes: nerve cell death in CNS.
- 3 ways to seperate proteins by CHARGE
- -Gel Electrophoresis
- 3 ways to seperate protein by SIZE
- SDS-PAGE electrophoresis
- 2 Ways to seperate proteins by BINDING
- -affinity chromatography
- Ion exchange chromatography
- -Column with beads (either - or +)
-Proteins bind to beads w/ opsit charge
-Proteins with same charge flow through
- 2 Types Ion Exchange Chromatography
- charged, binds + proteins.
+ charged, binds - proteins.
- Gel Electrophoresis (SDS PAGE)
- SDS - sodium dodecyl sulfate
PAGE - polyacrylamide gel electrophor.
SDS creates uniform negative charge.
Proteins put at top of acrylamide plate.
Migrate down, according to SIZE.
Smallest go fastest.
- sodium dodecyl sulfate
-in SDSPAGE, completely denatures all noncovalent (hydrogen) bonds in proteins - only primary sequence remains. Mercaptoethanol also reduces disulfide bonds.
- Reagents of SDS PAGE
- Limitation of SDS PAGE
- Completely denatures all bonds
- Isoelectric Focusing
- seperates proteins on basis of charge.
Mixture of AMPHOLYTES - many pI's.
Sets a pH gradient.
Add proteins; seperate as each seeks the pH of its pI.
- Trypsin and Chymotrypsin
-Key reagents in protein analysis
-Specifically cut proteins into fragments.
- hydrolizes peptide bonds after ARG LYS
- hydrolyzes peptide bonds after
PHE TYR TRP
- WHy use chymotrypsin and trypsin?
- Both cleave peptides after different residues; overlapping the cleaved fragments allows identification of similar bonds, to sequence the whole sequence.
- edman's reagent
- Edman's Reagent
-cleaves individual amino acids from the amino-terminal end.
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