protein structure Flashcards
hydrophobic amino acids
- alanine
- leucine
- isoleucine
- methionine
- phenylalanine
- valine
- glycine
- proline
DEFINE: tertiary structure
the way in which individual secondary structural elements pack together within a protein and between subdomains of a protein
how would you obtain primary structure when the dna and rna sequence is unknown?
sequence part of the protein using edman degradation then search databases to identify the rest of the protein
OR
mass spectrometry
Describe the process of Edman degradation
- add PITC to 1st AA
- acidify solution
- AA drops off due to PITC addition
- wash off AA from protein
- analyse AA using high performance liquid chromatography
- repeat
how would you obtain the secondary structure from the primary structure?
- de novo secondary structure prediction
- alignment of other proteins of known structure
- circular dichroism
describe methods of de novo secondary structure prediction
- biophysical modelling based on what is known about alpha helices and beta sheets
- software folding the protein, predicting if it is a high energy state or a low energy state and then choosing the protein with the lowest energy state
what is the far uv spectral region?
190-250nm
describe the process of circular dichroism
- take purified protein
- shine far uv, circularised light onto protein solution
- measure absorption
- calculate the fraction for each secondary structure type in a protein
what are some methods used to determine the 3d structure of a protein?
- x ray crystallography
- nmr
- electron microscopy
describe the process of x ray crystallography
- very large quantities of pure protein obtained
- concentrate protein to a very concentrated state - protein packs tightly together in an ordered structure to form a crystal
- fire x-rays through protein crystal
- deflected x rays give rise to diffraction pattern
- diffraction pattern captured on a piece of film
- trace back structure from diffraction pattern
nmr structure determination is an __________ process
iterative
DEFINE: proteomics
analysis of complete protein content in a living system, including alternatively spliced variants and post-translationally modified proteins
what is the 3 step protein purification in order?
- ion exchange
- size exclusion
- affinity
what is SDS?
negatively charged detergent causing proteins to unfold, giving it a more uniform charge
what is mercaptoethanol?
reducing agent breaking disulphide bridges between protein subunits
what factors affect protein migration in a gel during SDS-PAGE
- molecular mass
- amount of carbohydrate in protein
- phosphorylation in protein
what is isoelectric focusing?
separation of proteins by charge on a gel
describe the process of isoelectric focusing?
- sample is placed on a gel with a stable pH gradient
- proteins migrates in the electric field and stop when they reach isoelectric point = pH at which the protein has no net charge + stops migrating in the electric field
which amino acids can be phosphorylated and why can they be phosphorylated?
- serine
- threonine
- tyrosine
because they have -OH group
describe the process of mass spectrometry
- digest protein into peptides (using trypsin)
- ionisation of each peptide
- measure mass of each peptide
- separate ions by their m/z ratio
- detection of ions in proportion to their abundance
what is hydroxylation and what kind of enzyme is it performed by? provide an example.
hydroxylation: addition of an -OH to an amino acid
hydroxylases
e.g. adding -OH to proline —> hydroxyproline in collagen. defect = scurvy
