2: Proteins II Flashcards
What are the basic principles of protein purification?
Separate proteins based on differences in size, charge, binding affinity, solubility, to isolate target protein
What is gel-permeation chromatography?
A type of size-exclusion chromatography
Molecules separated by porous beads in the stationary phase
Larger molecules elute first because they cannot enter the pores and travel around them
Suitable for larger biomols
What is ion exchange chromatography?
Separates based on affinity for charged groups in the stationary phases
The stationary phase will have charged groups to attract the opposite charge, retaining the undesired charged molecule
Not effective for uncharged proteins
What is affinity chromatography?
Separating proteins based on their binding to ligands
Stationary phase functionalised with a ligand with a specific affinity for the target molecule
Highly specific
What is gel electrophoresis?
Separates protein by size
Smaller proteins move faster through the gel
Usually agarose gel for DNA or polyacrylamide for proteins
Use buffer solution, ions allow electricity to be conducted and maintain pH stability during electrophoresis
Molecules migrate towards electrode of opposite charge
Limited resolution for samples that are simliar in size
How can you estimate Mr using gel-permeation chromatography?
Compare elution volume of unknown protein to standards of known molecular weight
How does SDS PAGE estimate molecular weight?
SDS denatures the proteins
Proteins are then separated based on molecular weight
A protein’s migration distance is compared with known standards
How does ultracentrifugation help estimate Mr?
High-speed spinning used to measure sedimentation rate of proteins in solution
Rate depends on size, shape and density
Sedimentation patterns can reveal whether a protein exists as a monomer or in a multimeric form
How does mass spectrometry estimate Mr?
Mass to charge ratio (m/z)
Can also identify subunits and their stoichemistry in mutimeric proteins
How can these methods help determine quaternary structure?
Combining different techniques can help provide info on the size of the protein, the molecular weight, sedimentation behaviour, and exact masses
Comparing the Mr of intact proteins vs their subunits
This helps to deduce number and types of subunit
How do you measure protein concentration?
Using absorbance at 280nm
Proteins absorb UV light at 280nm due to aromatic amino acids
Absorbance at this wavelength is directly proportional to protein concentration
Using Beer Lambert Law
Protein conc = Abs / (extinction coefficient x length)
How do 2D gels show the proteome?
Separate proteins by isoelectric point and size to resolve complex mixtures
How can fluorescence detect conformational change?
Changes in environment of fluophores alter emission
Indicating structural shifts
What is circular dichroism and how does it show conformational change?
A spectroscopic technique used to study secondary structure by measuring difference in absorption of right-handed vs left-handed light by chiral molecules
CD measures these differences, reflecting secondary structure changes
How is 3D structure determined by X-ray diffraction?
X-ray diffraction determines 3D protein structure by directing X-rays at a crystallised protein
The X-rays are scattered by the atoms in the crystal, creating a diffraction pattern
This pattern is analysed to deduce electron density maps, which reveal the positions of atoms within the protein. The structure is then refined by fitting a model to these maps, allowing for the determination of the 3D arrangement of the protein’s atoms
How does NMR determine 3D structure?
Nuclear Magnetic Resonance (NMR) spectroscopy determines 3D protein structure by measuring the interactions between atomic nuclei, primarily hydrogen (protons), in a magnetic field
The technique detects the chemical shifts and coupling constants, which provide information about the distances and angles between atoms in the protein These data are then used to calculate the protein’s three-dimensional structure
What are binding and active sites in proteins?
Specific regions formed by 3D protein folding where ligands or substrates can bind
Often changes protein shape
What is a serine protease?
An enzyme that cleaves a peptide bond to release two peptides using serine’s hydroxyl group as the attacking group
What is the structure of haemoglobin?
Four subunits, tetramer
2 alpha, 2 beta
Each with a haem group containing Fe2+ that co-operatively binds oxygen
Histidine residue
How does the sickle-cell mutation affect haemoglobin?
Point mutation on beta globin gene in chromosome 11
Glu–>Val in the beta chain
Creates a sticky patch that causes aggregation in low O2 conditions
How do you approach determining the structure of a new protein like DNA polymerase?
Clone and express the gene
Purify the protein produced
Assess folding and function
Solve structure using X-ray/NMR
What is myoglobin?
A monomeric protein that primarily serves to transport and sore oxygen
Is only a single polypeptide chain and binds only one molecule of oxygen tightly
Releases little under physiological conditions
What are the T and R states of haemoglobin?
T (tense), deoxygenated
R (relaxed), oxygenated
What is the important amino acid residue for haemoglobin?
Histidine
Proximal histidine co-ordinates the Fe2+ in the haem
Distal histidine stabilises the oxygen binding site, helping to orient oxygen for binding
