MolBio2 - 45 Flashcards
Define K-off[AB]
Rate of dissociation of AB into A+B
Define K-on[A+B]
Rate of association of A+B into AB
Define protein interaction equilibrium
Association rate = dissociation rate; K-on = K-off
Define the protein interaction equilibrium constant
K-on / K-off
List 5 major methods of protein binding detection
ELISA, PAGE, Western Blot, Far Western Blot, Surface Plasmon Resonance
What does ELISA stand for?
Enzyme-linked immunosobrent agent
What does PAGE stand for?
Poly-acrylamide gel electrophoresis
What is surface plasmon resonance also known as?
Biacore
Draw how Biacore works
Describe how surface plasmon resonance works.
Bait molecule added to gold film by flexible tether, and inserted. Solution of prey molecules passes by, binding to some bait molecules. Binding causes measurable alterations in the plasmon resonance field.
What is plasmon resonance?
Magnetic field close to the gold strip, setup by incident light
What are the 2 key disadvantages to surface plasmon resonance?
Tether can block binding site, gold can affect the bait protein
List 4 methods of protein structure detection
Circular dichroism spectroscopy, x-ray crystallography, NMR, EM
What is circular dichoism?
Profile of the differences in absoprtion of left and right-handed polarised light
What does CD stand for?
Circular Dichroism
What spectral region of CD reveals protein secondary structure?
Far-UV - 190-250nm
What three types of protein secondary structure are there?
Beta-pleated sheet, alpha-helix and random coil
What does CD spectroscopy tell us about proteins?
% proportion of each kind of secondary structure, but no information on arrangement
What spectral region of CD reveals protein teriary structure?
Near-UV - 250-350nm
What particular signals are shown on teriary structure CD spectroscopy?
Aromatic amino acids and disulfide bonds
Why are aromatic acids and disulfide bonds particularly sensitive to CD spectroscopy?
Their absorbance is affected by the local environment and can be observed dynamically
What pre-requisites are there to x-ray crystallography?
Proteins need to be highly pure and in crystal form
Describe the mechnism of x-ray crystallography
High energy and focussed beam mof x-rays fired through protein crystal. Most pass straight through, some deflected giving diffraction pattern
How is the strucure of a protein determined from an x-ray crystallograph?
Structure traced back from diffraction pattern
What is the resolution of an x-ray crystallograph dependent on?
How high energy the x-ray is and how fine the detector is
What molecules are used in NMR?
H1, C13, N15
How does NMR work?
NMR active nuclei resonate at specific frequencies in magnetic fields. Dependent on local environment, different protons resonate at different frequencies - chemical shift
What is chemical shift?
Difference in proton resonation in NMR dependent on local environment
How are proteins generated for NMR?
Recombinantly in bacteria with only C13/N15 available, so they are NMR-active
Describe NMR structure results
Iterative process, so several possible structures are represented as an ensemble for analysis
How are specimens preserved in EM?
Negative stain or vitreous ice (cryo-EM)
What kinds of structure are best for EM?
More symmetrical and ordered leads to easier averaging process
What are the slowest protein structure analysis methods?
Crystallography - growing crystals; NMR - analysis
What are the quickest protein structure analysis methods?
Solution biochemistry or CD
What are the most expensive protein structure analysis methods?
Crystallography and NMR
What is the cheapest protein structure analysis method?
Solution biochemistry
What protein structure analysis method has the highest resolution?
Crystallography
What protein structure analysis method has the lowest resolution?
Solution biochemistry/CD - affinities/secondary structure only
What protein structure analysis methods have no limit on protein size?
Solution biochemistry, CD, crystallography
What is the limit on NMR protein sample sizes?
50kDa
What protein structure analysis methods require only small samples?
Solution biochem, CD, EM (to some extent)
What protein structure analysis methods require large sample sizes?
Crystallography, NMR
What are the possible problems with crystallography?
Can have crystallisation artefacts and dynamics analysis is limited
What are the disadvantages of NMR?
Need very high concentrations of sample AND isotopic labels, size limitation
What is a key limitation in EM analysis of proteins?
No dynamics
