Biochem 1-4 Flashcards
Macromolecules
Made of monomers or residues
Kinds: proteins polysaccharides nucleic acids lipids and membranes
Gibbs free energy change
deltaG = deltaH - TdeltaS
Protein purification techniques
1. Crystallize separate proteins from other molecules 2. column chromatography (interactions between matrix and proteins) ion exchange chromatography positive and negative charges Gel-filtrate porous matrix separate based on size Affinity covalently bound small molecule, separate based on interaction with the molecule
Saccharides
monomer. Also called a carbohydrate
carbon, hydrogen and oxygen
5-6 carbons
Fisher, Haworth, Envelope
Polysaccharides
covalently bonded saccharide monomers
glycosidic bond
acetal–two OR groups and two R groups on one carbon
dehydration reaction
Nucleic acids
(polynucleotides) composed of monomers called nucleotides: 1. 5 carbon sugar 2. heterocyclic nitrogen containing base 3. a phosphate or P containing group
ATP is an example
nucleic acid structure
In DNA and RNA nucleotides are connected via a 3’,5’ phosphodiester linkage
Lipids
rich in carbon and hydrogen few oxygen
simplest lipids are fatty acids
when combined with glycerol-3-phosphate they form glycerophospholipids which make up biological membranes!
Metabolism
2 parts: Catabolism breakdown to release energy Anabolism use energy to construct
Euks versus Proks
complex differentiated organisms versus single cell
proks have no nucleus rather nucleoid region
prok no internal membrane compartments euks have organelles
prok pili or flagella, high surface area to volume
Cytosol complexity
Stew of things!
Selectivity becomes important
highly organized
Biological functions of proteins
- enzymes
- storage, transport
- structural support
- mechanical work
- decode and regulate genetics
- hormones
- Abs, toxins and other fun things
Amino Acids
20 kinds (common ones)
amino group and a carby acid on the same carbon (called the alpha carbon)
R sidechain
chiral at alpha, some have extra chirality :D
Aminos and pH
at body pH (7.1 - 7.4): amino group protonated pKa 9, carby acid deprotonated pKa
Amino Stereochemistry
L-aminos are bae
a few D exist but they are rare
L is carby at top, amino is on the left
D is carby at top, amino on the right
Types of aminos
Aliphatic (hydrophobic) Aromatic (hydrophobic) Sulfur containing alcohol containing Basic Acidic
Aliphatic Aminos
Hydrophobic sidechains
Glycine [G] (Gly) exception! not very hydrophobic, also only AA with no chiral carbon.
Alanine [A] (Ala)
Valine [V] (Val)
Leucine [L] (Leu)
Isoleucine [I] (Iso) has second chiral center
Aliphatic Aminos with rings
My fav amino: Proline [P] (Pro).
Sidechain is cyclized on the alpha amino group (less nucleophilic)
pyrrolidine ring restricts geometry GETTIN’ KINKY
less hydrophobic than other aliphatic aminos
Aromatic Aminos
Phenylalanine [F] (Phe)
Tyrosine [Y] (Tyr) Can be ionized but not at body pH 280 nm
Tryptophan [W] (Trp) 280 nm
nm absorbance Can be used to find the conc of proteins in a solution
Sulfur containing aminos
Methionine [M] (Met) nonpolar methyl thioether
Cysteine [C] (Cys) dimerize to form cystine pKa = 8.4
Alcoholic Aminos
Serine [S] (Ser)
Threonine [T] (Thr) second chiral center
uncharged polar side chains with beta hydroxyl groups
weakly ionizable pKa ~16
nucleophiles (especially in active sites)
Basic Aminos
Histidine [H] (His)
Lysine [K] (Lys)
Arginine [R] (Arg)
nitrogenous bases
at body pH they are protonated and polar
histidine pKa is near body at 6–often transiently protonated, used in active sites
Acidic Aminos
Aspartate [D] (Asp)
Glutamate [E] (Glu)
Aspargine [N] (Asn)
Glutamine [Q] (Gln)
asp and glu have carby acids in the sidechain
deprotonated at body pH, negative charge
asn and gln are primary amide derivatives of the other two–highly polar but uncharged
Biosynthetic Aminos
More than 200!
sometimes made in biological pathways
employ decarboxylation and deamination enzymes:
adrenaline, thyroxine
sometimes chemically modded once inside a protein
Ionization of Aminos
2 Pkas–the one from the carby and also the amino
Ionization influences:
3D shape of proteins
enzyme catalysis
Henderson-Hasselbalch
pH = pKa + log[A]/[HA]
Amino Titration
Cation–>zwitterion–>Anion
Isoelectric Point
The pH where a molecule is electronically neutral
Amino Titration Histidine
Special because side chain can be ionized
3 pKas
Peptide bond
linear sequence is called the primary structure
amino acids are linked through an amide bond also called the peptide bond
dehydration reaction
N terminus to C terminus
Peptide nomenclature
amino residues change their –ine or –ate to –yl
glutamine becomes glutamyl
name from N to C
Sodium Dodecylsulfate Polyacrylamimde Gel Electrophoresis
SDS-PAGE
separate small mixes of proteins
migration in electric field
SDS detergent overwhelms the native charge on a protein
Mass spectrometry
determine mol weight and amino sequence in protein s
-Electrospray Ionization (ESI) and MAtrix assisted desorption ionization (MALDI) MALDI can be used to find post transcriptional mods as well
Percent composition of proteins
Acid hydrolysis followed by PITC treatment and detection
Edman degradation procedure
identity of each a.a. starting from the N terminus
- treat with PITC at pH 9
- Treat PTC peptide with anhydrous acid like TFA
- extract anilinothiazoline product, treat with aqueous acid to make phenylthiohydantoin derivative
- identify with chromatography
- repeat sequentially
Puts limits on the number of sequential aminos you can detect
Cyanogen Bromide BrCN
cleaves polypeptide chains on the C terminus side of methionine residues
Proteases
Trypsin catalyzes cleavage on the C terminus side of Lys and Arg
Chymotrypsin catalyzes cleavage on C terminus side of aromatic hydrophobs like Phe, Trp, Tyr
Polypeptide sequencing
- treat with hydrolytic enzymes
- analyze fragments via Edman degradation
- deduce structure
Proteomics
study of large sets of proteins
native conformation
a protein’s natural shape at physiological pH
Four levels of protein structure
1. primary linear structure of aminos 2. Secondary regularities due to H bonding and other interactions 3. Tertiary folded and compacted polypeptide chain. interactions between secondary structure 4. Quaternary Domain interactions
Primary
linear sequence along the polypeptide backbone
Secondary
alpha helix
beta sheet
Tertiary
interactions between secondary. Side chains in helix to helix or sheet to sheet
Quaternary
Domains of the protein interacting
3D structure depictions
Space filling models–radii to illustrate overall shape and surface
Ribbon structure–simplifies backbone and shows secondary structure
Ball and Stick–highly detailed show H bonding and other molecular interactions
NMR
used to determine protein structure
Proteins do not have static immovable structures!
Peptide bond conformations
phi N–Ca bond
trans or cis. restricted for proline
psi Ca–C bond
trans and cis
+ is clockwise
- is counter
the Carby–N bond doesn’t rotate very much. Its called omega
Ramachandran Plot
Way to tell where the bonds can be rotated/are sterically permissible
alpha helix or beta sheet is what is liked
Alpha Helix
right or left handed
Pitch–how many nm peptide advances per turn
Rise–nm advance per a.a.
3.6 AA residues, 13 atoms per turn so 3.6 sub 13 helix
psi and phi for the bond angles
side chains oriented outward
a.a sequence confers stability
Ala is fine. Tyr or Asn not fine. Gly destabilizes. Pro won’t allow for H bonding
FACIAL DIRECTIONALITY
Beta Sheet
Parallel–R groups line up on top and bottom
Anti-parallel perfectly straight bonds, the R groups and the h alternate
Right hand twist but mostly flat
side chains above and below
technically is a 2 and 3 structure
more flexibility in bond angles
Amphiphatic
Loops and Turns
Loops usually have hydrophilic residues at or near surface
~10% of Aminos
short loops called turns (
Common motifs
- helix-loop-helix
- coiled coil
- helix bundle
- beta-alpha-beta
- hairpin
- beta meander
- greek key
- beta sandwich
Protein Domain
25-300+ AA residues
covalently connected to other domains by noncovalent interactions
Common domain folds
different functions in protein
- enzymatic activities
- surface recognition elements
- ligand binding
Quaternary structure
- subunits in an oligomeric protein have a set stoichiometry
- greek letters to describe subunits
- weak covalent reactions hold subunits
- (4 structure creates active site)
Protein Denaturation
- Altering the native conformation will result in denaturation
- loss of normal activity
- energy can be small to accomplish this
- many ways to denature–heat and chemicals
- characteristic mel temp (50-60 C)
Chemical denaturation
- Disrupt the hydrophobic interactions
- Chaotrophic (urea and guanidinium salts)
- Detergents
Denaturation and Disulfide
Example: Native ribonuclease + urea and beta mercaptoethanol denatures the protein, reduces disulfide bonds. Take away ME randomly reforms bonds . Some small proteins can reform
Protein Folding
- can be assisted by molecular chaperones!
- uses n ATP
example: heat shock proteins
Case Study: Collagen
- connective tissue and structural protein
- molecule of collagen contains three left-handed helical polypeptide chains coiled around each other to form right-handed supercoil
- inter-chain hydrogen bonds
- polypeptide sequence has pattern Gly-X-Y
- X is often Pro
- Y is often modified Pro
- high proline content makes collagen rigid
Case Study: Collagen
- Individual collagen triple-helices are crosslinked via Schiff bases
- Allysine residues are chemically-modified from Lys
Case Study: Antibodies
- Ab are integral to the immune system
- Abs recognize epitopes on antigens
- most abundant are the immunoglobulin G (IgG) class.
- tetramers with two heavy chains and a light chain.
- linked by disulfide bridge. heavy chains have 4 domains and light have 2 domains.
- Common motif: immunoglobulin fold–sandwich of two antiparallel beta sheets
- high affinity for antigen. Heavy chain is specific to organism of origin
