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Transcript
Protein NMR terminology
COSY- Correlation spectroscopy
Gives experimental details of interaction between hydrogens connected
via a covalent bond
H
H
H
H
C
C
C
N
NOESY- Nuclear Overhauser effect spectroscopy
Gives peaks between pairs of hydrogen atoms near in space (1.5-5 Å)
(and not necessarily sequence)
CH
H
H
N
Fingerprint region
1
2
dH
TOCSY
Walk
along
the
sequence
gH
bH
3
4
5
COSY
9.0
NH
O
C
7.0
NOE
CH3
N
C
C
H
H
O
NOE
COO
CH2
H
H
O
N
C
C
CH2
Ala
CH
H3C
-
CH3
N
H
H
7.0
Connectivites by NOE
dN - Connects CH of residue i to NH of i+1
dbN - Connects CbH of resdiue i to NH of i+1
dNN - Connects NH of residue i to NH of i+1
LEU
dN
H
dN
dNN
N
dbN
H
ALA
VAL
C
dNN
C
H3C
H
N
CH3
CH d O
bN
C
C
H
N
C
H
CH3
dNN
O
H
dN
CH2
dbN
Hi-NHi+3
Hi-NHi+1
i+3
H
i+2
An -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
N
i+4 the sequence (sequential nOes
H
connect neighbouring residues)
NOE
H
For an -helix we see Hi-NHi+3
and Hi-NHi+4 nOes.
i
Assignment of secondary structural segments
• sequential stretches of residues with consistent secondary
structure characteristics provide a reliable indication of the
location of these structural segments
A b-strand is distinguished by strong CHi-NHi+1contacts and
long range nOes connecting the strands.
A long range nOe connects residues more than 5 residues apart
in the chain.
A real example.
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??
Summary of the Sequential and Secondary NOEs observed for
rat FAS ACP - most definitely structured
NHi-NHi+1
iNHi+1
biNHi+1
GDGEAQRDLVKAVAHILGIRDLAGINLDSSLADLGLDSLMGVEVR
D
D
D
DD
D
D
D
NHi-NHi+2
Hi-NHi+2
Hi-NHi+3
Hi-NHi+4
Hi-bHi+3
CSI
J
0-00000---------+-0-0--0+--+0+---+00+-0-----+ ++ -------+--+++++ +++-+++ --- ----- --QILEREHDLVLPIREVRQLTLRKLQEMSSKAGSDTELAAPKSKN
NHi-NHi+1
iNHi+1
biNHi+1
D
D
D
D
DD D
D
DDD
NHi-NHi+2
Hi-NHi+2
Hi-NHi+3
Hi-NHi+4
Hi-bHi+3
CSI
J
-----0+-+0++--0--00+--------00000000+0+00-00
-++-+++ - -- -+-+ - -+- +++++++
+++
So I have assigned the NMR spectrum and connected the
amino acids. I have a good idea of the secondary structure.
What next??
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!). O
H
N
CH
C
O
H
N
CH 2
Asn
C
O
CH
C
Gly
H
N
H
H
OH
Identified as an
asparagine aminohydrogen from COSY
spectra
HO
O
C
CH 2
Glu
H2C
C
O
CH
NH 2
NOE indicated the
asparagine amino-hydrogen
is near a glutamate acidic
hydrogen
Schematic showing long range nOes in the lac headpiece protein
What next? STRUCTURE CALCULATIONS
•From NOE I know close atom-atom distances, but that doesn’t give a structure
•The information you have up to this stage is a list of distance constraints
•The structure can be determined by inputting this information to computer
minimization software.
•The computer program also contains information about amino acids, bond
lengths/angles and standard information about atom-atom interactions such as
minimum distance (i.e. Van der Waals radii)
•With all this information you can generate a model of the structure.
Important: NMR gives you a number of possible solutions
(all almost identical, rmsd <1Å), This can range from 5-20 models
X-ray crystallography give one average structure
NMR structures can be averaged to give one average structure as well
Excerpt from an NOE table for Actinorhodin Polyketide ACP - 1997
This file contained ~ 700 lines of nOe restraints
! Thr7 NH
assign (resid
7 and name HN
)(resid
75 and name HD1* ) 4.0 2.2 0.5
assign (resid
7 and name HN
)(resid
75 and name HD2* ) 4.0 2.2 0.5
assign (resid
10 and name HN
)(resid
75 and name HD2* ) 5.0 3.2 0.5
assign (resid
10 and name HN
)(resid
75 and name HD1* ) 3.3 1.5 1.0
assign (resid
72 and name HN
)(resid
31 and name HD1* ) 5.0 5.0 0.5
assign (resid
72 and name HN
)(resid
31 and name HD2* ) 3.3 1.5 0.5
assign (resid
72 and name HN
)(resid
31 and name HB*
) 4.0 4.0 1.5
assign (resid
72 and name HN
)(resid
31 and name HA
) 4.0 4.0 1.0
75 and name HN
)(resid
10 and name HD1* ) 4.5 4.5 1.0
! Leu10 NH
!Arg72 NH
! Leu 75 NH
assign (resid
The simulated annealing protocol - begin by simulating a 1000K
heat bath and generate an extended model strand
Start
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
30 ps
Start to cool the system and increase the penalty for bad contacts.
20 ps
Minimize the final structure to see if it satisfies all the nOes
A simulated annealing trajectory over the first few picoseconds
4 helices begin to
‘condense’
Unfolded
Correctly folded
Challenges for Interpreting
3D Structures
• To correctly represent a structure (not a
model), the uncertainty in each atomic
coordinate must be shown
• Polypeptides are dynamic and therefore
occupy more than one conformation
– Which is the biologically relevant one?
Representation of Structure
Conformational Ensemble
Neither crystal nor
solution structures
can be properly
represented by a
single conformation
 Intrinsic motions
 Imperfect data
Uncertainty
RMSD of the ensemble
Representations of 3D
Structures
C
N
These 2D methods work for proteins up to about 100 amino acids,
and even here, anything from 50-100 amino acids is difficult.
We need to reduce the complexity of these 2D spectra.
1
16
1
H
O
HN
12
12
C
14
N
R2
14
N
C
16
O
12
C
12
C
1
R1
1
H
HN
We can start by
replacing 14N with
15N, a spin 1/2
nucleus.
Run a ‘COSY’ type experiment that correlates an amide proton
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
15N.
So how well dispersed are the 15N shifts? Is it worth trying to separate
our spectra out based on their differences?
1H-15N
HSQC of rat FAS ACP
Why?
•The more we understand about a protein and its function, the more we can do
with it. It can be used for a new specific purpose or even be redesigned too
carry out new useful functions (biotechnology & industry).
•We can use this knowledge to help understand the basis of diseases and to
design new drugs (medicine & drug design).
•The more knowledge we have how proteins behave in general, the more we
can apply it to others (protein families etc)
A case study - Leukocyte function associated protein-1 (LFA-1)
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
mechanism
Inflammation (chronic) is responsible for asthma and arthritis.
Developed small molecule inhibitors and test binding
O-
O
O
N
S
A
B
C
N
N
D
N
O
Weak binding
mM to mM
see a migration of the peaks
A more successful inhibitor- nM ‘tight’ binding.
See unbound and bound
populations
Solve NMR structure of complex…
Helix 7 is
prevented from
shifting
NMR is a diverse tool with which we can study protein structure.
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.
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.