topic 03 protein structure & analysis Flashcards
when and between what is the peptide bond formed?
between alpha-amino group of one amino acid and the alpha-carboxyl group of another in a condensation reaction
how are peptides written?
N-terminal first.
Gly-ser does not equal Ser-gly
what is the repeating pattern of a peptide?
NCC
NCC-NCC-NCC
even though peptide bond is a single bond, why does it shares characteristics with a double bond? what is steric interference?
due to the resonance between the C-N and C-O bonds
the atoms are coplanar and there is no free rotation around the C-N axis which constrains flexibility and could prevent some folding patterns. this is steric interference
define primary, secondary, tertiary, and quaternary structure in general terms
primary: sequence of amino acid residues
secondary: local folding pattern of polypeptide backbone
tertiary: 3D structure of entire polypeptide chain including side chains
quaternary: arrangement of polypeptide chains in a protein with multiple subunits
what are the bonds involved in the primary structure?
covalent bonds between the residues
what are the bonds involved in the secondary structure?
hydrogen bonds between the backbone NH and CO groups
in an alpha-helix, a carbonyl is hydrogen bonded to an amine, 4 residues away
in a beta-sheet, bonds formed between neighbouring beta strands
between a parallel and non-parallel beta-strand, which one is stronger? why? why is the other not as strong?
the non-parallel beta-strand:
more stable bc of the straight hydrogen bonding
the parallel beta-strand is not as strong due to the bends and kinks
what are the bonds involved in the tertiary structure?
all 4 forces: ionic, hydrogen, disulfide, and van der waal
between side chains or side chain and backbone (distant in the sequence)
what are the bonds involved in the quaternary structure?
all 4 forces: ionic, hydrogen, disulfide, and van der waal
what are the two major secondary structures?
alpha-helix and beta-strand
which amino acids cause destabilizing kinks in the alpha-helix? why?
proline
it can’t fit into into the backbone’s pattern and it has an imidazole ring that constrains bond angles
glycine
too flexible. can disrupt secondary structures
how are side chains position in an alpha-helix? beta-sheet?
alpha-helix: protrude out
beta-sheet: up and down
what does the folding of a secondary structure depend on?
interaction between side chains
steric interference from large side chains
charge repulsion between side chains
presence of proline/glycine
what is the most important force in the folding of tertiary structures?
entropy & the hydrophobic effect
entropy:
when folding, hydrophobic area comes together in protein’s interior, expelling water. even though the peptide’s entropy decreases, as it goes from unfolded to folded, overall entropy increases because of the water’s entropy. when unfolded, the water in the protein shields the hydrophobic parts from the rest of the water. when folded, water is released into the solution and returned to high entropy state
the hydrophobic effect:
proteins are most stable when hydrophobic residues are in the core and hydrophilic on the surface
describe the disulfide bond. where can it be found? why?
covalent bond between cysteine residues (their thiol groups)
only present in (oxidized environments) non-cytoplasmic proteins bc in cytoplasmic proteins, there exists enzyme systems which remove the disulfide bonds
give two examples of quaternary protein structures
collagen: triple helix made from 3 polypeptides
elastin: made of individual elastin peptides cross linked
why is a folded protein considered its native state?
said to be the conformation with the least free energy
what denatures a protein?
treatments with solvents that weaken bonds
extreme pH
high temperatures
describe a denatured protein
has a random, flexible conformation
usually lacks biological activity
may aggregate or precipitate due to exposed hydrophobic groups
if the denaturing condition is removed, some proteins will refold and regain activity. what does this renaturation tell us?
that all the info for folding is within the primary structure
describe molecular chaperones
proteins that help others fold and renature
they mask hydrophobic areas to prevent aggregation during the process - play important role in newly synthesized proteins
they can also act as a chamber to provide the protein with the right environment
define conformation changes
movement of domains connected by flexible linkers
describe a domain
independently folded part of the domain that folds into stable structures
a protein could have one or more domain
a domain is in between secondary and tertiary structure
have structural and functional purposes
what are domains derived from?
primordial proteins that existed on their own, that’s how they can fold independently
what separates domains?
separated by loosely folded regions and may create clefts between them
describe a protein family
proteins related by evolution
they have similar primary sequence, functions, structure, and domains
give examples of protein families and describe them
lipoproteins: combined with lipids
metalloproteins: combined with proteins
glycoproteins: modified by attachment of carbohydrates
hemoproteins: attached heme-group
describe a conserved residue
amino acid residue that’s necessary for function and could be found in domains
describe membrane proteins
inserted or tightly bounded into the membrane
define proteases
enzymes that break down proteins
define homologous proteins
have the same protein family
what are the purification steps of a protein
- cell breakage
- centrifugation for initial fractionation
- column chromatography to separate proteins from one another
- small samples of the preparations are monitored at different stages of by SDS (sodium dodecylsulfate) polyacrylamide gel electrophoresis (SDS-PAGE)
describe SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoresis, the most common analytical separation technique
separates by the polypeptide chain size
charged molecule migrates in electric field
gel acts as a molecule sieve: smaller molecules go faster
the gel can be a single concentration or a gradient. gradients are used to examine a range of different protein sizes
a low % gel is used for high molecular weighed proteins
what are the different types of liquid chromatography and what differences do they exploit to separate them?
gel filtration: size
ion exchange chromatography: net charge
affinity chromatography: presence of binding affinity for a specific ligand
what techniques can be used to determine 3D structures?
x-ray (crystallography) diffraction or NMR (nuclear magnetic resonance)
cyro-EM (cyro-electron microscopy) can be used to solve structures of large proteins and protein complexes that are difficult to obtain in large amounts and to crystallize
describe column chromatography
contains insoluble beads with different characteristics
applies proteins in solution where they will pass through and around - some will emerge from the bottom where they’re collected and separated due to the time it takes to go through. a “fraction” is collected every few minutes
what is effluent?
discharged liquid collected in fractions - every few minutes
how does one tell which collected fraction (effluent) contains proteins?
using the absorbance at 200-280 nm
aromatic amino acids absorb at 280nm
other amino acids at 200nm due to carboxyl group
describe gel filtration chromatography
the beads contain holes
if a protein gets into the hole, it is slowed down - otherwise, it will come out early. this is an example of “size exclusion”.
smaller proteins fit into the bead more easily, therefore, larger proteins are more likely to elute faster
describe ion exchange chromatography
relies on attraction of opposite charges
to remove proteins that are stuck on sides, increase salt concentration or change the pH
define isoelectric point (PI)
pH at which a specific molecule carries no net electrical charge
if pH < PI, there’s a positive charge
describe affinity chromatography
relies on interaction between protein and a specific ligand
if the ligand is attached to a column matrix, certain proteins will bind to it and unbound proteins are washed away by the buffer
most powerful chromatography method but needs the knowledge of what ligand to use
protein of interest is eluted by adding competing free ligand, or by changing the pH which changes the ionization of the group responsible for binding
what is polyacrylamide?
long chain of hydrophilic polymer
the gel in SDS-PAGE is polyacrylamide
what does the SDS in SDS-PAGE do?
denatures proteins and give a negative charge
this must be done before electrophoresis
beta-mercaptoethanol added to break disulfide bonds
differentiate between old and new technological methods of analysis
old tech
- purify proteins, then sequence (chemical method)
- identify genes, then deduce amino acid sequence
new tech
- use mass spectrometry to characterize proteins in complex mixture (proteomes)
- can provide info on identity (sequence), abundance, and various modifications
what can mass spectrometry measure?
molecule size
mass/charge ratio
whether it’s protonated or not
what are the steps of mass spectrometry?
now, steps 1-3 can be forgone
- extract protein
- run on SDS gel
- cut out desired band
- resolved by liquid chromatography. before this step, sample is treated with an enzyme to fragment the proteins into manageable bits
LC-MS
5. sample run in mass spectrometer 2
6. mass determination
LC-MS/MS
5. mass spectrometer
6. collision cell
7. mass spectrometer 2
8. amino acid sequence identification
describe x-ray crystallography
obtain a highly pure protein, then get it to form a crystal by concentrating protein solution so much - then blast it with x-rays. a pattern will be caught on the detector due to the diffraction
diffraction pattern of crystal = diffraction pattern of molecules
the diffraction pattern yields an electron density map
describe NMR (nuclear magnetic resonance)
can determine the structure of the solution directly (in the solution, the protein has more conformational freedom)
the environment of the nucleus affects the magnetic resonance
generates a 2D NMR for 2 different nuclei in protein (H & a heavy isotope)
atm, NMR can only be used for small proteins
describe and list steps of cryo-EM
cryo-electron microscopy. used for larger proteins
- vitrify solution: preserve proteins in native solution state
- electron microscopy: imaging electron density to build model of structure - image one single molecule at a time
