CHEN EXAM 1 Flashcards
C-C single bond length
1.5 A
C=C double bond length
1.3-1.4 A
hydrogen bond length in biosystem
2-4 A
optimum van der waals distance
4.5 A
hydrophobic interactions
powered by the increase of entropy in water (favors increase in entropy)
how can you deprotonate dsDNA?
heat or increase pH, pH 9.9 most strands denatured (thymine), at 9.7 about half (guanine)
down syndrome
trisomy 21, 3 copies of chromosome 21 due to chromosome segregation error
TATA-box Binding Protein (TBP)
molecular structure conserved among archebaterium, plants, and humans
amino acid ionization states
both protonated (in acidic conditions): NH3+ and COOH
zwitterionic: NH3+ and COO-
both deprotonated (in basic conditions): NH2 and COO-
hydrophobic amino acids
GAPLIVYMWF
guanine, alanine, proline, leucine, isoleucine, valine, tyrosine, methionine, tryptophan, phenylalanine
polar amino acids
STYNQC
serine, threonine, tyrosine, asparagine, glutamine, cysteine
basic amino acids
KRH
lysine, argenine, histidine
acidic amino acids
DE
aspartic acid, glutamic acid
histidine
acts like a physiological pH sensor, pKa=6
can accept or donate protons at this pH
cysteine cross-linking
oxidation can induce disulfide bonds between two cysteines
occurs in insulin
sickle cell
substitution of valine for glutamic acid
change in primary structure affects function
survived due to malaria advantage
cis/trans configurations of amino acids
based on if the alpha carbons are on the same side (split by peptide bond)
trans is lower energy and favored
proline configurations
cis and trans have similar steric clashes so both isomers exist, isomerase can convert the proline from one configuration to another
C-N peptide bond length
1.32 A
C=N bond length
1.27 A
C-N single bond length
1.49 A
which angles in amino acids can be rotated and which are fixed?
peptide bond (C-N) is fixed due to partial double bond character/resonance
alpha C and NH3 (phi) can rotate, -80
alpha C and COO (psi) can rotate, 85
ramachandran plot
illustrates favorable and possible torsion angles
torsion angles
phi and psi angles in amino acids
how many DIFFERENT proteins are there in 1 human cell?
10,000 - 30,000
levels of protein structure
1: AA N to C sequence
2: a helix or beta sheet via hydrogen bonding
3: 3D folding via side chain interactions (noncovalent bonds, Cys disulfide bonds)
4: interaction of tertiary structures of 2+ polypeptide chains
alpha helix characteristics
right handed coil with protruding R groups
CO and NH groups H bond
3.6 residues/turn (3.6 AA/turn)
1.5 A/residues-rise (height of one AA)
5.4 A/turn-pitch (height of one turn)
beta sheets
distance between adjacent AAs: 3.5 A
antiparallel or parallel or mixed
antiparallel arises by hairpin folding of a single strand
twisted sheets, hydrophobic/hydrophilic faces
how can primary structures predict secondary structures?
identifying amphipathic helix (alpha helix has both polar and non polar)
beta strands have hydrophilic and hydrophobic faces
chameleon sequences
can adopt alpha helix or beta strand in different contexts
reserve/beta/hairpin turn
C-O group of residue i is H-bonded to the N-H group of residue i+3
polypeptide loops
part of antibody molecule
lie on protein surfaces
participate in interactions between proteins
protein motifs
common combinations of secondary structure present in many proteins, usually similar function
ex. helix turn helix in DNA binding proteins
coiled-coils
two right-handed alpha helices intertwine and form a left-handed super helix
stabilized by ionic and VDWs
heptad repeat & leucine zipper
3.5 residues per turn
allows side chain pattern to repeat every 7 AAs
2 helices interact through leucine side chain
collagen
rich in gly and pro
3 helical polypeptide chains form a superhelical cable
substitution mutation of Gly –> osteogenesis imperfecta
hemoglobin structure
2 alpha-globin, 2 B-globin subunits = heterotetramer
icosahedron
example of quarternary structure, 4 subunits of 60 each
many viruses: rhinovirus, coronavirus
denaturing proteins
urea & B-mercaptoethanol
bring a denatured protein back to its native form
remove agents, dialysis, oxidation
amyloidoses
diseases resulting from protein aggregates (amyloid fibrils)
Alzheimer’s, Mad Cow Disease
beta sheets are prone to aggregate
one aggregate is a nucleus to recruit more
green fluorescent protein (GFP)
from jellyfish, can be attached to a protein to determine cellular location
oxidation of SYG polypeptide chain = modification for fluorescence
proteome
entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions
gradient centrifugation
separate molecules based density
protein purification
homogenate supernatants undergo a series of centrifuges, the final supernatant is the cytoplasm of soluble proteins
salting out
solubility of proteins vary with salt concentration
when salt conc increases, different proteins precipitate out
dialysis
removes salt from protein solution
dialysis bag holes let salt equilibrate but do not let protein through
gel-filtration chromatography
separates protein by size
smaller proteins enter beads
larger proteins exit first
ion-exchange chromatography
separation of proteins by charge
beads are charged, same charged proteins will leave and opposite charged will bind
bound proteins released by increasing salt conc. or adjusting buffer pH
affinity chromatography
beads with attached target chemicals will bind proteins with high affinity to chemical
bound proteins are released by passing the target chemical through the solution
high-performance liquid chromatography
very fine beads + high pressure pumps move liquid through column, increased interaction sites (surface area) > higher resolving power
SDS PAGE
coat proteins in negative charge, separate in gel electrophoresis by size
stain proteins to visualize
isoelectric focusing
separates protein by pI value
protein is put through a pH gradient and goes to the pH where it has no charge
2D gel electrophoresis
1) isoelectric focusing and 2) SDS electrophoresis
separates by charge (pH) then size, each unique
antibody diversification
hypermutation of antigen binding site
monoclonal antibody
binds to 1 epitope
polyclonal antibody
binds to different epitopes on the same antigen
hybridoma technology
beta cells and cancer cells form immortalized B cells and can induce tumors
ELISA (enzyme-linked immunosorbent assay)
quantifies amount of protein present based on color
indirect: antigen, antibody, then second antibody linked to fluorophore
sandwich: antibody, antigen, antibody
western blotting / immunoblotting
SDS PAGE
transfer to polymer
stain with primary antibody specific to protein
stain with 2nd antibody specific to primary antibody
2nd antibody attached to fluorescence
co-immunoprecipitation
antibody incubation + antibody binding protein –> complex can be separated and analyzed
mass spectrometry
protein sample is ionized by a laser
electrical field accelerates ions toward detector
lightest ions arrive first, time of flight for ions is measured
edman degradation
protein exposed to PTH which reacts with N-terminus, last AA can be released without cleavage, identify the AA at N-terminas
X-Ray crystallography
detects x-ray diffraction
only 1 formation (limitation)
determines exact atomic location
NMR
detects nuclear spin states
nuclei of atoms absorb different frequencies called chemical shifts
smaller proteins only, approximate (not super accurate)
nuclear overhauser effect
identifies pairs of protons in close proximity (adjacent hydrogens)
cyro-electron microscopy
detects electron density
larger proteins, exact atomic location
RNA viruses
RNA genome, mRNA to make protein
retrovirus
RNA > DNA > RNA > protein
nucleoside
base and sugar
nucleotide
base, sugar, phosphate
2 ‘OH
2 ‘OH in RNA can be deprotonated in basic conditions (auto-hydrolysis), unstable
B DNA structure
R-handed, 3.4 A rise, 10.4 bp/turn, 20 A diameter, 35.4 pitch, C2’ endo sugar pucker
chargaff’s rule
A=T and C=G but A+T does not equal C+G
hammerhead ribozyme
first discovery of enzyme made of RNA
catalytic RNA that can catalyze bond hydrolysis
tyrosine pKa
10
cysteine pKa
8.3
lysine pKa
10.4
argenine pKa
12.5
aspartic and glutamic acid pka
4.1
pitch
height of a turn, rise * (residues/turn)
rise
height of a residue, A/residue
pKa of N terminal
8
pKa of C terminal
3.1
structure of A DNA
R-handed, 2.3 A rise, 11 bp/turn, 26 A diameter, 25.3 pitch, C3’ endo sugar pucker
structure of Z DNA
L-handed, 3.8 A rise, 12 bp/turn, 18 A diameter, 45.6 pitch
major and minor grooves length
13 and 9 A
how many base pairs in human genome?
6.4 B
how long is DNA per cell?
2 m
DNA base length
1.2 nm
Angstrom to nm
10 A = 1nm
A N1 pKa
4.5
C N3 pKa
4.2
what % of DNA codes for proteins?
1
cell nucleus diameter
10 μm
