BCH-Week 5 Flashcards
what is the idea behind basic spectroscopy
the protein that was purified will be hit with beam of light and the wavelengths it absorbs will be detected and tell us something about the structure
What are two types of spectroscopy that are focused on secondary structure of protein
IR spectroscopy which uses infrared light
CD spectroscopy which uses circulary polarized light
What is fluoresecnce spectroscopy mainly used for
to localize structural determination
What are some methods that we can use to look into the 3D structure of protein
X ray spectroscopy, NMR, Cryo-EM, and alphafold
What does the IR spectroscopy read and tell us
IR spectroscopy is based on protein being hit with infrared light and then the absorbation will be measured which depends on the vibration in the bond
depending on the frequency, the contents in the secondary structure can be known
what is the range in IR spectroscopy that tells us the structure contain alpha helix, beta sheet, random coil, or beta turn
alpha helix- 1650 to 1660
beta sheets- 1620 to 1640
turn/ antiparallel beta sheets- 1675 to 1695
random coil- 1640 to 1650
What does CD spectroscopy base its readings on and what does it tell about teh structure
CD spectroscopy uses circulary polarized UV light and the chiral alpha carbon and secondary structures will absorb one direction of light over the other
What numbers are on the readings from CD spectroscopy are associated with alpha helix, beta sheet, and random coil
alpha helix- 222 nm and 208 nm reading negative while 195 nm reading positive
beta sheets- 217 nm reading negative
random coil- 198 nm reading negative
What is advantage of using CD spectroscopy over IR spectroscopy
CD spectrosocpy will tells us about the protein’s prefered environement as it requires sampel to be put in a solution and that solution’s environment can be changed to know how protein reacts in them to know about protein’s hydrophobicity, polarity, etc
IR spectrosocopy is also useful for when comparing protein structure varying and causing diseases, like the case for prions
How does X ray crystallography work to produce pure crystal of proteins
the protein will be crystalized once purified and concentrated using teh right environment and that crystal can be hit with beam of X ray which will be diffracted by teh elctron clouds around teh atoms in protein and that will localize the atom’s positions and help us create an image for teh structure
How does NMR spectroscopy work and what are some of its advantages
NMR is carried out with the protein in solution so that means it can be used to monitor interactions and modifications of the protein
it is based on nuclear spin and that will be meaured by hitting teh solution with strong electromagentic field and measure the absorption to deduce teh nucleus’s environment and determine protein structure
How does CRyo electron microscopy work
the sampel will be prepared on fine grid and frozen, then it is put in electron microscope which measures teh beam of electron passing through protein and that diffraction will be used to determien the stucture where images at different positions will be captured and then put together to find the protein’s image
What are chromophores
functional groups that contain double bonds which absorb UV light and visible light at specific wavelengths
Where can chromophores be found in proteins
in the aromatic rings and amide carbonyls
How can presecence of chromophores be used to determine protein’s structure
structure of protein will influence accessibility of these chromophores to light absorption and be used to characterize protein’s structure
How are fluorophores useful
fluorophores will absorb and emit light known as fluorescense which can be used for cellular localization
How does tryptophan fluorescence work and what can it tell us of protein’s environment
tryptophan fluorescence work having the indole ring of trptophan abosorb UV light at 270-295 nm and then will emit it somewhere between 310-355 nm and these wavelenghts will depend on polarity of its enviroenment
red shifted- longer wavelengths so solvent is exposed, similiar polarity
blue shifted- shorter wavelengths so solvent is burried, different polarity
What is fluorescence microscopy useful for
will allow us to see the protein in cellular context but instead of fusion proteins that only show where proteins are, using fluorescently tagged antibodies like FRET and BRET will also help detecting distance dependant protein protein interactions
What are soem techniques used to look into protein protein interaction, protein molecule interactions
for protein protein interactions, these methods can be used: coimmunopercipitation and pull down assay, cross linking reagents, and FRET
for protein molecule interactions: transport assay and enzymatic reaction
How does pull down assay help to know if one protein is interacting with another
by pulling down the protein of interest and looking if any other proteins come along with it as well using antibodies specific for protein of interest and antibody specific for the other protein that might get pulled down
where antibody for protein of interest will be used first to be able to seperate for that protein, then using Western Blot, we will look for the other protein, if it is detected by binding the antibodies specific for it, then there is interaction
What is the idea behind chemical cross linking
it consist of covalent bond forming between molecules
what is the distinct characteristic of BioID
a technique that will allow for identification of many protein peotein interaction in cell at once, it uses enzyme called BirA which is a biotin protein ligase that fuses to protein of interest and will bionylate any close proximity peoteins
the bionylated proteins will be purified using streptavidin beads and identified using WB or mass spec
