Lecture 12 - 16 Flashcards
Advantages of NMR over X-ray Crystallography
Protein can be in solution - natural state
Can study protein dynamics
Can see interactions that protein makes
Atoms used
1H, 13C, 15N - spin of 1/2
Halpha chemical shift in secondary structure
Lower for alpha helix than beta sheet
How to determine alpha helix/beta sheet from chemical shift index
4 or more consecutive -1/+1
J
spin-spin coupling constant - when nearby nuclei split a signal
Relaxation
Time (T) taken for NMR signal to disappear - shorter T means there are other nuclei nearby
Nuclear Overhauser Effect (NOE)
Depends on relaxation between spins - if you saturate one nuclei, the nuclei nearby will have a stronger signal
R
Distance between 2 relaxing nuclei
NOE signal + distance between nuclei
Strong - 1.8 - 2.7A
Medium - 2.7 - 3.5A
Weak - 3.5 - 5A
Amide protection
When amide protons are involved in hydrogen bonds in a folded protein, they are protected from exchange with solvent and exchange more slowly
Protonation factor
Rate of expected change (unfolded)/ Rate of actual exchange
Problems with large molecule NMR
Issues with unresolved signals Overlapping signals Complex multiplets Low sensitivity - use high B0 Large quantity of info - use computational methods
Ligand
Non-macromolecules that interact with proteins - water not normally counted as ligand e.g. peptides nucleotides, ions
Surface characteristics of protein
Size, shape, charge, hydrophobicity, dynamics e.g. flexibility
Most favourable protein shape
Globular/spherical
Protein crystallisation steps
Purify protein Crystallise protein Collect data Evaluate data Model building 3D structure
Why is it hard to crystallise proteins?
Proteins not evolved to crystallise, may be dynamic, every protein requires different parameters, can be unstable
Structural proteins
Keratin, collagen, fibroin
Motor proteins
Actin/myosin, kinesin, ATP synthase
Fibroin
Antiparallel beta sheets with ala and gly side chains
To get pure spider silk
Ligate consensus sequence into expression vector, transform into e.coli and express and purify
MotA/MotB
convert proton gradient into energy for movement
FliG, FliM and FliN
Interact with MotA/B to cause a direction change
FliF
self-assembled, forms template for rest of structure to assemble from
Amino acid in outlier range in ramachandran plot
Glycine - so small that doesn’t really have many steric clashes
Protein homeostasis maintained by…
protein folding/unfolding
Folding is exothermic/endothermic
exothermic
Half unfolding point
melting point - Tm. sharp change between folded and unfolded state due to cooperative binding
Excreted misfolded proteins
Aggregate, which can form amyloids - result in disease
Promiscuous activity
duplicated gene has a small degree of secondary activity , this can become the main activity if beneficial through mutation.
Glycosyl hydrolases involved in..
Degredation of poly/oligosaccherides
Saccherification
hydrolysis into soluble sugar
Hydrophobic cluster analysis
shows folding similarities through hydrophobic/hydrophillic residues - can determine structural families
Digestive protease
Low specificity, works well in acidic environment
General proteases
high specificity, only cleave at one specific peptide bond in one specific molecule. adapted to specific environment
How do mutations make drug ineffective?
Change in sequence, change in structure, change in binding site = ligand/drug can no longer bind
