The Advantages Of Paramagnetic NMR

169 Pages·2013·2.34 MB·English

by  Florian Siepel

The Advantages Of Paramagnetic NMR
Dissertation
fortheaward ofthedegree
Doctorrerum naturalium
oftheUniversityofGöttingen
withinthedoctoralprogram PhysicsofBiologicaland ComplexSystems
oftheGeorg-AugustUniversitySchool ofScience(GAUSS)
submittedby
Florian Siepel
from Neustadta. Rbge
Göttingen2013
Thesis committee
Prof. Dr. ChristianGriesinger
NMRbased Structural Biology,Max Planck InstituteforBiophysicalChemistry,Göttingen
Prof. Dr. Jörg Enderlein
III. InstituteofPhysics,UniversityofGöttingen
Prof. Dr. Andreas Janshoff
InstituteforPhysicalChemistry,UniversityofGöttingen
Members ofthe ExaminationBoard
Referee:
Prof. Dr. ChristianGriesinger
NMRbased Structural Biology,Max Planck InstituteforBiophysicalChemistry,Göttingen
2nd Referee:
Prof. Dr. Jörg Enderlein
III. InstituteofPhysics,UniversityofGöttingen
Further Members oftheExaminationBoard
Prof. Dr. Andreas Janshoff
InstituteforPhysicalChemistry,UniversityofGöttingen
Prof. Dr. MarinaBennati
Electron SpinResonance Spectroscopy,Max Planck InstituteforBiophysicalChemistry,Göttingen
Dr. ClaudiaHöbartner
NucleicAcid Chemistry,Max Planck InstituteforBiophysicalChemistry,Göttingen
Dr. AdamLange
NMRbased Structural Biology,Max Planck InstituteforBiophysicalChemistry,Göttingen
Dateoforal examination: 28.10.2013
Affidavit
I hereby declare that I wrote this thesis The Advantages Of Paramagnetic NMR on my own and
withouttheuseofanyotherthanthecited sourcesand tools.
...........................................................................................
Place, Date Signature

Climb,climb, climb!

VII
Acknowledgments
First of all, I’d like to thank my doctoral advisor Prof. Griesinger who gave me the oppertunity and
the freedom to work on two interesting but different topics. He was always contactable and had a
quick andinformativeanswerto allofmymanyquestions.
I would also like to thank my thesis committee members, Prof. Enderlein and Prof. Janshoff. Their
input in the committeemeetings was of great value and led to very useful discussions and develope-
ments.
I thank the other members of the examination board, Prof. Bennati, Dr. Höbartner and Dr. Lange,
that theyarewillingtojoinmyoral examination.
Thanks to Dr. Andrei Leonov whose knowledge about chemistry and his always granted help had a
hugeimpactonthiswork. IthankKerstinOverkampandGerhardWolfformanyHPLCpurifications
of theDNA samples and the measurements of the mass spectra. Thanks to Dr. Dirk Bockelmann for
all thehelpwiththecomputersandsoftware.
My specialsthankstoall readers ofthisthesis,especiallyEibeDücker.
Thanks for the great atmosphere in the lab to Sebastian Täubert, Eibe Dücker, Manuel Schmidt, Dr.
NinaSchützenmeister,Dr. FabianPeters and RuthLinder.
Philip Lottmann, Sebastian Täubert, Dr. Saskia Villinger, Dr. David Ban, Dr. Donghan Lee, Dr.
Pablo TrigoMourinoand MartaCarneiro: Thankyou foralltheusefuldiscussionsofall kind.
ThankstoallmembersoftheZ-roundandthewholedepartmentfortheniceandfriendlyatmosphere.
VIII
I’dliketoespeciallythankSaskia,PhilipandÖkkiforthegreatfriendship,thetimeIspentwitheach
ofyou andall thethingswe’vedoneandexperienced.
EinRiesendankgehtanmeineEltern,GroßelternundSchwester,diemichnachanfänglichemZögern
doch immeraufmeinemzweiten Bildungswegrestlosunterstützthaben.
IX
Summary
There are three distinct effects in nuclear magnetic resonance spectroscopy (NMR) that differ be-
tween paramagneticanddiamagneticmoleculesinisotropicsolution. Theseare residualdipolarcou-
pling(RDC),pseudocontactshift(PCS)andparamagneticrelaxationenhancement(PRE).Allofthese
effects are dependent on intermolecular angles and distances for a given nucleus of interest and can
provide information about the structure and dynamics of a molecule. In order to obtain this valuable
information, the molecule is required to display paramagnetic characteristics. One of the up-to-date
methods achieves this requirement via the use of small molecular tags that coordinate paramagnetic
metal ions. Most of these tags are attached to a protein via a disulfide bridge formed with a solvent
exposed cysteine residue. Thus, in order to use this technique for DNA, new tagging strategies are
required.
In this work, a modified nucleobase was synthesized allowing introduction of a sulfur moiety into
the DNA. This provides a method to attach any tag that is based on a disulfidebridge, directly to the
SH
O
N
N
HN
N
DMTO O N
O
O
HN
O
O P DNA O O N
NC N O
O
DNA
Left: The modified nucleobase containing a carbon triple bond. Right: The final modification of the
DNAprovidingasulfurmoiety.
X
DNA.WiththisnucleobaseacarbontriplebondisintroducedintotheDNAstrand,andasubsequent
cycloadditionreaction leads tothefreesulfurmoiety.
The modified nucleobase was successfully tested by tagging a self-complementary DNA strand
(24 nucleotides), in which the modified nucleobase was introduced during the DNA synthesis. The
Cys-Ph-TAHA tag, preloaded with lutetium, terbium or thulium, was attached via a disulfide bond
resulting in a tagged DNA strand loaded with a lanthanideion. However, even with this milestone, a
major aspect of this work was to develop a reliable and reproducible purification and sample prepa-
ration protocol. This became a critical element, sincethe tagging of DNA as compared to proteins is
challengingby theabilityforthephosphatebackboneto coordinatelanthanideions.
In the theoretical framework section, a complete step-by-step derivation of the three major para-
magneticeffectsstartingfrom first principlesis given. Forthederivationoftheequationsdescribing
theRDCs,PCSsandPREs,expressionsforthedipolarHamiltonians,crossrelaxationrates,alignment
induced RDCs, correlation functionsand spectral densitiesare presented.
The second topic of this work is based on a different paramagnetic effect. In order to over-
come the lower sensitivity of NMR compared to other spectroscopic methods, there are many re-
ports on approaches that increase the polarization of the investigated nuclei, i.e. that create hyper-
polarized species. One of these methods, photochemically induced dynamic nuclear polarization
(photo CIDNP), is based on short lived radicals created by direct illumination of the sample in the
magnet with a laser beam. Within the scope of this thesis, a photo CIDNP setup was planned, built
andtested. Thefirstexperimentsandresultswithtriethylenediamine,tyrosineand3-fluoro-L-tyrosine
demonstrated the usefulness as well as the limitations of this technique. For 3-fluoro-L-tyrosine a
complete analysis of the relaxation behaviour, including cross relaxation and cross-correlated relax-
ation, isgiven.