FINAL EXAM Flashcards
4 MAJOR CLASSES OF BIOMOLECULES
nucleic acids
proteins
carbohydrates
lipids
Lipids are different from proteins, polysaccharides and nucleic acids in that:
They aren’t polymers of monomers. They are an association of similar units.
Biochem connects genotype and phenotype through various ‘omes’
genome -> transcriptome -> proteome -> metabolome
What are each of these?
Genome = protein coding human genes Transcriptome = collection of mRNA Proteome = collection of proteins Metabolome = breakdown and synthesis of biomolecules.
Anabolism
biosynthesis of larger biomolecules
Catabolism
degradation (oxidation) of molecules
Reactions move toward order/disorder
disorder
For a reaction to be spontaneous, deltaG should be +/-ve
deltaH should be +/-ve
detlaS should be +/-ve
-ve
-ve
+ve
The major chemical elements in living system
C, N, O, P, H, S
Chemical conformation is defined by
rotation around covalent bonds
Chemical configuration changes require
making and breaking bonds.
What is D?
D = dialectic constant = force between two ions D = 0 in a vacuum and D = 80 in water
Vander waals interactions
Very weak attractive forces doe to dipole or induced dipole interactions of closely spaced atoms.
Atoms can’t get closer than their radii without covalent bonding.
Hydrogen bonds are strongest when
3 atoms are in a line
Hydrogen bonds are
when one hydrogen atom is shared between two electronegative atoms. Partial covalent character
Hydrogen bonding between two peptide groups would be strongest….
at protein surface exposed to water (D=80)
Hydrophobic Effect
Since water is polar it repels the non polar substance, losing entropy.
So, when a non polar substance is placed in water it will congregate to decrease the amount of SA exposed to water.
Leads to clusters, beading, micelles/vesicles/bilayers
If an acetate buffer (pH -4.76) solution is at pH 5.76, what is the ration of [CH2COO-] to [CH3COOH]?
10 : 1 because one cahnge in pH up means that there is 10 times as much base as acid int here.
What thing affects the usefulness of the buffer?
pK value and total concentration
Why does DNA have a polar exterior?
Negative phosphate groups
Most common form of DNA
B form
Things that stabilize the DNA double helix?
hydrogen bonds
Mg2+ binding to phosphate backbone
hydrophobic effect
Oligonucletotides…
are short stretches of nulceotide polymer that will hybridize with matching ssDNA.
PROBES!
Exons make up how much of our total DNA?
ABout 2%
miRNA
regulates gene expression by blocking mRNA translation or stability
siRNA
work similarly to miRNAs. Have therapeutic potential.
Dominant level of regulation of gene expression in prokaryotes and eukaryotes
initiation of transcription
IF a genome, proteome and metabolome are defines by the number of genes, proteins and metabolites, what’s the order?
Proteome > genome > metabolome
isoelectric point
Every amino acid has an isoelectric point - the pH value at which, the net charge of the molecule is 0
Low number for acid AAs and high number for basic AAs (intuitively)
Proline
The only imino acid. The side chain connects to the NH group and forces a kink into the polypeptide.
Trypotophan
is the largest AA
Glycine
Is the only achiral AA
Cysteine
Has a sulhydryl group that can be oxidized to forma disulfide bond with another cysteine. Inside the cell it’s usually in SH form and outside the cell it is usually in S-S form. It can be kept in SH form invitro by adding reducing agents (mercaptoethanol or diotheitol)
Acidic AAs
Aspartate and Glutamate
Basic AAs
Lysine and Arginine
How should be write proteins in conventional form
N->C direction
Protein conformational diseases
ALzheimers, Prions (CJD, Mad Cow)
Alzheimers
Transition between a-helix and B-sheet problem
Prions diseases
CJD and Mad Cow
Protien undergoes conformational change that changes its function. Change to B-sheet allows stacking of it into a fibre
Protein Motifs
Small regions with a defined stequence or structure, often serving a common function in different proteins
Protein Domain
Sub regions of single poly-peptide chains that can fold and function independently
Proteins can evolve by swapping one domain for another (eg antibodies)
wireframe model
shows every atom
Backbone model
shows only the polypeptide backbone shape
Ribbon model
shows the secondary structures
Spacefill model
Shows the vander waals radii
Denaturation
conformational change that leads to loss of function
The native state
functional state of a protein
The native state is stabilized by
- Burying NP side chains
- Optimizing H-bonding
- Maximizing side chain packaging in interior (vander waals)
Denatured state is favoured by
- Increased conformational entropy of the unfold R group
- heat, chemicals, pH extremes, mutations
Chaperonin assisted folding of proteins
While the primary protein structure dictates its 3D structure, many larger proteins require assistance to explore their conformational space before something bad happens to them
Anfisen Experiment
Native structure of RNAse only recovered is urea is removed before/during removal of the reducing agent, but not after
Levinthal Paradox
If explored randomly it would take the age of a universe to explore all of the conformations possible for a protein, but they do it in seconds.
Folding is directed by rapid formation of secondary structure that limits further possibilities.
Some regions of proteins are intrinsically disordered. What does this mean?
They only fold upon interaction with binding partners.
Dialysis
A semipermeable sac and small molecules (urea, mercaptoethanol) may leave while large molecules (RNAse) stay.
Differential Centrifugation
- a type of subcellular fractionation
- Done in buffer w similar density to water
- Initially low speed; big things to bottom
- Centrifuge supernatant at higher speed to get second pellet of organelles
- Keep spinning at higher and high speeds for ribosomes and large molecules
Isopyonic centrifugation
Gradient of density where lower density things are at the top. Spin until light things migrate to top and heavy things are at bottom.
The most discriminating property of a protein is…
its function
Affinity chromatography
Protein will bind to an immobilized, specific ligand and all other proteins will pass through. Use an affinity tag to the recombinant protein for expression and purification. BInds to the protein and extracts it.
Size exclusion chromatography
Packed course polymer beads and run protein and solvent through. Large proteins will come out first.
Ion exchange chromatography
Polymer beads with -vely charged functional groups. Protein mixture added and basic proteins won’t ionize until higher pH
Proteins with -ve charge will move out first with -ve beads.
Adding salt interacts with the -ve beads and displaces the proteins.
SDS-PAGE
reducing agent usually added to cleave S-S bonds and resolve individual polypeptide chains
2D page
Separates based on pH and size.
Can be useful for revealing disease related to protein levels.
Protein sequence techniques (3)
1) Mass Spec
2) X-Ray Crystallography
3) Protein NMR
Mass Spectrometry
A way to deduce protein sequence.
Distinguish proteins on a basis of mass to charge ratio.
Trypsin used to cleave Lys-Arg
Get Protein Mass Fingerprinting - each pattern has a unique pattern of Lys/Arg Fragments
Tandem MS-MS further breaks up the fragments at peptide bonds and finds masses of fragments on a coupled second mass spectrometer.
X-Ray crystallography
3D arrangement of atoms in a protein deduced by measuring diffraction of xrays in a protein crystal.
Provides detailed information on conformation, enzyme mechanism and binding.
Protein Data Bank stores deduced structures.
Protein NMR
Samples the nuclear environment using spin
Electromagnetic resonance requency of nuclei in a stron magnetic field can prvide information on neighbouring atoms
Done in solution so can give info on mobility (see frays on the end where there is more space for movement)
Post Translational modifications
Phosphorylation
Glycosylation
Proteolysis
Phosphorylation of Proteins
Transfer a PO4 group from ATP. Catalyzed by protein kinases.
Regulates metabolism and signal transduction
Glycosylation of proteins
Attachment of carbohydrate group to Asn and Ser on secreted, EC or luminal proteins
Proteolysis of protein
A way to synthesize peptide hormones.
Ubiquitin and proteasomes
The final post-translational modification
Ubiquitinate and degrade proteins
Invariant residues on hemoglobin and myoglobin
The proteins are different but the things they have in similar indicate critical functions.
Hemoglobin and myoglobin curves
Hemoglobin has a sigmoidal curve meaning it can release much more oxygen at lower pO2
Myogolib oxygen binding is hyperbolic.
Myoglobin strcture
monomeric. Oxygen binds to the Fe2+ porphyrin heme prosthetic group. When the heme is buried, it can’t be oxidized.
Hemoglobin ALlosteric effectors
H+ (Bohr Effect) - CO2 production in tissues leads to decreased pH, protonating a side chain and stabilizing T state
CO2 rects with N terminal to form carbamate. Stabilizes T
BPG binds at the +vely charged hole, ONLY in the T state to stabilize it. Increased at high altitudes and in chronic anemia (favours O2 release)
Sickle Cell anemia
Point mutation in DNA codes for structurally different hemoglobin. It polymerizes into long fragments and distrorts the RBC
IgG
2 heavy chains and 2 light chains stabilized by intra and inter-chain disulfide bonds
Organized into complement and antigen binding regions of globular B-sheet domains
Actin filaments
- Microfilaments (smallest)
- Fibrous, F actin is a polymer of globular, G actin
- +ve pointed end and -ve barbed end
- Organized into cables by actin binding protein
- Functions: cortical skeleton under p. membrane and motility.
F-Acting Assembly and Treadmilling
ATP bound to G actin hydrolyzed when it is incorporated into F-actin
Polymerization more rapid at +ve end.
Capping proteins block +ve end polymerization.
Severing Proteins break filaments.
Hydrolysis of ATP allows treadmilling. Net poly at +ve end and loss at -ve end.
phalloidin
fungal compound that stabilizes F actin - too much assembly
cytochalasins
fungal compound that destabilizes f actin
Immunochemical detection
- F(AB) has heavy and light chain residues
- Specific binding to antigen’s EPITOPE due to complementary surface, H bonds and electrostatic interactions
- Antibody specificity can be used to detect selected proteins by ELISA and Western Blot
Collagen
Fibrous. Most abundant protein in human body.
Twisted braid of 3 extended chains. Uses proline for kinks.
Makes cables and meshes in EC matrix, connective tissue and bone.
GLYCINE at every 3rd residue allows proximity of chains.
Interchain hydrogen bonding strengthened by hydroxyglycine and hydroxyproline residues.
Diseases of collagen
Scurvy (requires ascorbing acid for hydroxylase rxn)
Ehlers-Danlos (stretchy skin)
Osteogenesis imperfects (brittle bone)
Elastin
Fibrous protein.
Deformable. Found in elastic tissues.
Chains connected by Lys-cross linking
Cleaved in alveolar wall by ELASTASE, which is inhibited by a-1 antitrypsin, which has decreased activity in EMPHYSEMA
Where does collagen processing occur?
ER, Golgli and outside of the cell. Further assembly outside of the cell.
Microtubules
Reversible aggregates of tubulin heterodimer forming hollow tubes.
Polar - polymerize and depolymerize from +ve end. Regulated by GTP.
-ve end anchored to an organizing centre (radiate and retract).
Straight and rigid tracks, form cilia and flagella.
Target of anti-cancer drugs.
Which filament is the target of anti-cancer drugs
Microtubules
Intermediate filaments
Insoluble fibrous proteins.
Fundamental unit is a a-helical coiled coil., strengthened by NP residues bt helices.
No polarity
In nuclear lamin, keratin in epithelial cells and neurofilaments.
Must depolymerize during mitsosis (nuclear membrane)
Actomyosin
ATP hydrolysis generates muscle contraction.
Thick and thin filaments slide past each other because of myosin.
Myosin head bound to an actin subunit of a thin filament. ATP binds to myosin head and it releases actin. Hydrolysis of ATP causes rotation of myosin and rebinding of actin farther along.
Binding actin causes ADP + P to be released from myosin and myosin head returns to normal conformation. (rigor)
3 ways to incr rate of rxn
increase temp
increase [ ]
add catalyst
How do enzymes do what they do?
Put substrates in proximity
Put them in correc torientation
Provide functional groups for catalysis
Carbonic anhydrase
One of the fastest enzymes converts CO2 to H2CO3 in the blood to regulate pH of the blood.
3 types of enzyme catalysis
acid base
covalent
metal
Acid-base catalysis
Transfer or removal of H+ lowers free energy of the transition state
Covalent catalysis
Transient formation of a covalent bond bt enzyme and substrate (enzyme acts as a nucleophile, donating an electron pair)
Metal catalysis
Metal plays a direct or indirect role.
At the active site, metal ions fan mediate redox reactions, promote reactivity of other functional groups or interact directly with the substrate.
Ex: Alcohol dehydrogenase uses a Zn ion to stabilize the developing O-
apo enzymes
don’t require cofactor
holoenzyme
enzyme with a cofactor for the reaction
The elctrophile
is an electron deficient atom
Chymotrypsin
A member of the serine protease family with a preference for bulky, NP residues on the carboxyl side of the peptide bond.
Uses covalent and acid-catalyzed catalysis in hydrolysis of a peptide bond.
Catalytic triad of Asp102, His57 and Ser195 at the active site of all serine proteases.
Flexible protein. Adopts diff conformation as it goes through catalytic cycle - induced fit.
Catalytic triad
Asp102, His57, Ser 195 on all Ser Proteases.
Asp102 anchors His57
His57 acts as a base and later as an acid
Ser195 acts as a nucleophile to attack the peptide bond.
Replacing Asp102 or His57 has a huge -ve effect, but replacing Ser195 isn’t as bad.
Serine Proteases
Syntehsized as inactive precursors (zymogens) and are activated by cleavage by other proteases (autoactivation)
COX isozymes
Catalyze the 1st step in the synth of prostaglandins
COX 1 involved in prostaglandin production and COX2 does pain and inflammation.
Both cox isozymes are targets of NSAIDs
COX 2 has a larger active site so there are now COX2 specific drugs.
Glucogenic
AAs that can be converted into pyruvate, oxaloacetate or other citric acid cycle intermediates
Ketogenic
AAs that can only be converted into acetyl CoA, ketone bodies or FAs
