Paramagnetic Relaxation Enhancement

Data analysis and structure calculation

Bernd Simon
NMR facility manager
EMBL Heidelberg
 Overview

NMR of paramagnetic samples

Sample preparation and Measurement of PREs
Data evaluation and calculation procedures
Implementation in ARIA/CNS

Literature:

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF.

Mackereth CD, Madl T, Bonnal S, Simon B, Zanier K, Gasch A, Rybin V, Valcrcel J, Sattler M.
Nature. 2011 Jul 13;475(7356):408-11.

An efficient protocol for NMR-spectroscopy-based structure determination of protein complexes in solution.

Simon B, Madl T, Mackereth CD, Nilges M, Sattler M.
Angew Chem Int Ed Engl. 2010 Mar 8;49(11):1967-70

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 Paramagnetic Relaxation Enhancement
e
Rtotal Ri RSpin
i
Rspin Rpara Rdia PRE
N
N CC PRE = Rpara - Rdia
N Stable Radicals
N Nitroxide, O2
H Metal ions
H
Ti3+, V3+/2+, Mn3+/2+, Fe3+/2+
Lanthanides

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 Solomon-Blombergen
Dipolar interaction electron-proton

JSB= c /1+( c)
2
c =( r + s )
-1 -1 -1

Paramagnetic effects depend on properties of electron

spin anisotropy and electron relaxation

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 Paramagnetic properties

Figure: Pintacuda et al., Acc. Chem. Res., 40 (2007) 206-212.

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 PRE PCS RDC

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 PRE and dynamics

N
N
rbond
H <r> 1 1 1
H e
c s r

1 1 1 1
t i s r
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 Cloning strategies

Remove all accessible native Cys

Check accessibility with Ellmans reagent
Structure should not be altered by mutation
Cys free mutant might be useful for crystalization
Introduce single Cys at selected sites
Conservative mutations
Amino acid solvent accessible
Rigid backbone structure preferred
Scan the whole surface
Design more mutants than needed

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 Spin labelling
1. Prepare protein sample in non reducing buffer:
If Ni-column then last 3 washes and elution without reducing agent
2. Measure protein concentration
3. Calculate amount of spin label you need
4. Add methanol to the dry spin label pellet
5. Add more than nearly equal molar amounts to the protein
6. Place reaction tube in dark with alu and incubate 2-12 hrs
7. Exchange in suitable buffer (NMR buffer) to get rid also of free spin label (see PD10).

Reduction with ascorbic acid:

0.5 M ascorbic acid - ca. 100 mg/ml
Chemicals:
3-(2-Iodoactamideo)-proxyl Sigma 253421- 2 ul in 500 ul NMR sample = 10 mM
50mg
MW: 325.17
Dissolve in 2.5 ml methanol
100 ul each in 1.5 ml eppendorf tubes
Go in speed vac and evaporate methanol
Store 2 mg batches in dark tubes (alu) in
freezer. Recent Review: Paramagnetic tagging for protein structure and dynamics
analysis. Keizers and Ubbink Prog. NMR Spectr. (2011)

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 Site Specific Paramagnetic Labelling

Protein-SH

Paramagnetic Diamagnetic

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 Calculation Protocol
RRM1
Step 1
input domain structures
PDB, homology model, etc.

RRM2
confirm backbone conformation
local refinement if necessary
chemcial shifts, RDCs, dihedrals, H-bonds

Step 4
structure of complex

Step 2 water refinement

linker randomization

global domain orientation

RDCs, cluster analysis

Step 3 distance restraints

PREs, spin label calibration

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 ARIA1.2 /CNS

ARIA: python-based, structure calculations with CNS

We use a single iteration (option already calibrated
data)
Modifications are only in the cns-scripts:
patch CYS residues to attach (multible) nitroxides
generate template with starting structures
retain structures with flexible restraints (ncs)
Introduce PREs as distances restraints

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 Spinlabels and Structures

Define amino acid sequence and patch the CYS-SL

protocols/generate.inp
patch csl reference=nil=(residue ###) end
patch csl reference=nil=(residue ###) end

Define input structure and randomized regions

run.cns
ncs1_choice: NO (no ncs-force on residues)
All (ncs-force on all atoms of residues)
ncs1_random: YES (phi-psi randomization of residues before simulated annealing)
NO (no randomization)
ncs1_start: first residue in group
ncs1_stop: last residue in group
ncs1_force: force constant for ncs-restraints of this group (0-10000)
ncs1_pdb: name of pdb-file with coordinates for the residues in run1/data/sequence

Check template structure especially the Spinlabels

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 Add four copies of
spinlabel to each cystein
side--chain
side 10 different samples
with single Cys

On average 18
Refine position of distances per spinlabel
spinlabels using intra
domain distances only
Distance constraints +/-+/-
2 A or +/-
+/- 0.2 ratio error

Calibrate distances check

for variation of c (grid
search or standard)

final c = 4 ns
Calculate structure using
interdomain PREs

Remove18/07/2013
spinlabel,,
spinlabel
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generate wt sequence,
water refinement
 Template Structure
SER281

ALA318
ASP273 Are all spinlabels there are
they solvent exposed?
ASN155
ALA164
Are the input structures o.k.?
ALA287
ALA171

THR209
ALA188

LEU187

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 Calibration of distances from PRE data

Peak intensity ratio of oxidized and reduced form of the

same sample
Battiste & Wagner Biochemistry (2000)

I para exp R2PRE 1 1 3

R dia R2PRE S(S 1) 2
H g2 2
B 6
4 c
c
2 2
I dia
2
R2dia R2PRE 15 r 1 H c

Initial distance calibration: linear Ipar/Idia

approximation for 0.2 < Iox/Ired < 0.8
Estimate of correlation time
Estimate of diamagnetic T2

Python scripts:
InitialDist_from_ratio_R2_tauc.py
correlation_time.py
GetHN_T2.py Distance r []

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 Acknowledgements

Proton relaxation is generally multiexponential

Diamagnetic T1 and T2 are not uniform
PRE correlation time from 1 and 2 as usual:
c = H
-1 * (3 1/2 2-7/4)1/2
Curie-Spin relaxation (only if r and c are significantly
different) -> field dependant
Mobility of spinlabel might be site-dependant (grid-search
for c ) Tobias Madl
Cameron Mackereth
Katia Zanier
Alexander Gasch
Michael Nilges
Michael Sattler

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