Protein NMR terminology. COSY- Correlation spectroscopy Gives experimental details of interaction between hydrogens connected via a covalent bond NOESY- Nuclear Overhauser effect spectroscopy Gives peaks between pairs of hydrogen atoms near in space (1.5-5 Å )
COSY- Correlation spectroscopy
Gives experimental details of interaction between hydrogens connected via a covalent bond
NOESY- Nuclear Overhauser effect spectroscopy
Gives peaks between pairs of hydrogen atoms near in space (1.5-5 Å)
(and not necessarily sequence)
An a-helix can be recognised
by repeating patterns of short
range nOes. A short range nOe
is defined as a contact between
residues less than five apart in
the sequence (sequential nOes
connect neighbouring residues)
For an a-helix we see aHi-NHi+3
and aHi-NHi+4 nOes.
A b-strand is distinguished by strong CaHi-NHi+1contacts and
long range nOes connecting the strands.
A long range nOe connects residues more than 5 residues apart
in the chain.
The rat fatty acid acyl carrier
protein. Involved in fatty acid
biosynthesis and part of a
larger subunit, the synthase,
Is it structured by itself??
rat FAS ACP - most definitely structured
So I have assigned the NMR spectrum and connected the amino acids. I have a good idea of the secondary structure.
At this point we notice there are still many nOes we have not assigned on the 2D spectrum. These are neither sequential or short range. They are long-range and connect residues more more than 5 amino acids apart (But still close in space!).
NOE indicated the asparagine amino-hydrogen is near a glutamate acidic hydrogen
Identified as an asparagine amino-hydrogen from COSY spectra
Schematic showing long range nOes in the acids. I have a good idea of the secondary structure.lac headpiece protein
What next? acids. I have a good idea of the secondary structure.STRUCTURE CALCULATIONS
This file contained ~ 700 lines of nOe restraints
heat bath and generate an extended model strand
Apply the distance restraints from the NOE data (perhaps 1000
restraints for a protein of 90 amino acids). Weight the nOes to
favour the formation of local secondary structure and later long
range structure. Allow chain to move through itself
Start to cool the system and increase the penalty for bad contacts.
Minimize the final structure to see if it satisfies all the nOes
A simulated annealing trajectory over 1000Kthe first few picoseconds
4 helices begin to
Neither crystal nor solution structures can be properly represented by a single conformation
RMSD of the ensemble
NRepresentations of 3D Structures
and even here, anything from 50-100 amino acids is difficult.
We need to reduce the complexity of these 2D spectra.
We can start by replacing 14N with 15N, a spin 1/2 nucleus.
with the 15N nuclei.
This is a heteronuclear experiment, I.e. we are looking at two
different nuclei, a 1H and a 15N nucleus. The ‘COSY’ type
experiment is beyond the scope of these lectures but is known
as HSQC, or heteronuclear single quantum coherence spectroscopy.
This refers to how the magnetisation is transferred from the 1H to the
So how well dispersed are the 15N shifts? Is it worth trying to separate
our spectra out based on their differences?
1 protonH-15N HSQC of rat FAS ACP
This protein is involved in tethering a leukocyte to a endothelium,
allowing migration through the tissue to a site of inflammation.
One domain of LFA-1, the I-domain is 181 amino acids and
undergoes a conformational change where helix 7 slides down the
protein, switching it into an active open form. This open form
is competent for cell surface binding.
If we can stop this switch, we may have an anti-inflammatory
Inflammation (chronic) is responsible for asthma and arthritis.
Weak binding (LFA-1)
mM to mM
see a migration of the peaks
See unbound and bound
Solve NMR structure of complex… (LFA-1)
Helix 7 is
It gives us information in solution under ‘physiological’ conditions
2D and 3D techniques combined with modern assignment methods
have allowed proteins up to 40 kDa to be solved.
The power of NMR lies not just with its ability to solve structures
but also its ability to probe binding of ligands and partner proteins
in ‘real’ time.
Many aspects we have not had time to deal with. NMR reveals how
proteins move in solution - can see domains flexing with different
timescale motions. These often correlate with binding patches
on the protein.
Textbook I recommend reading. structure.
J Evans - Biomolecular NMR Spectroscopy.
Chapter 4. Protein Structure, pages 147-174. After p174
numerous examples of NMR structures, labelling etc.
Chapter 2. More high level NMR approach - description
of how pulse sequences (I.e. COSY, TOCSY, HNCA etc) work.
Beyond the scope of the course but may be of interest.
Chapter 3. Details of calculations - for you details not important
but will give you more of an idea of how we use the NMR data to
calculate the structure.