The Ph.D School of Science Post-translational modifications of an archaea esterase & rapid

158 Pages·2017·19.19 MB·English

The Ph.D School of Science
Post-translational modifications of an archaea esterase &
rapid analysis and acquisition of NMR relaxation
dispersion data
December 2017
Ph.D. dissertation of:
Troels Schwarz-Linnet
Advisors:
Associate Professor Kaare Teilum &
Associate Professor Qunxin She
This thesis has been submitted to the PhD School of The Faculty of Science,
University of Copenhagen
SBiNLab
Structural Biology and NMR Laboratory
Department of Biology, Ole Maaløesvej 5
2200 Copenhagen N, Denmark
December 2017
i
Preface
This thesis describes an overview of 2 main projects, which have I pursued
duringmyPhDworkatSBiNLab,CopenhagenUniversity. ThePhDperiodwas
from February 2012 to August 2015, where i subsequently pursued a position
as high school chemistry teacher at Gentofte HF. During the two years as
chemistry teacher, 2 articles was published related to the two projects and this
thesis was written from summer 2017 until completion in December 2017.
The two projects was very diverse in their scope of theory and practical
work. Chapter one is concerned about post-translational modifications of an
archaeaesterase. Thisprojectincludedarangeoflaboratoryexperimentswithin
structural biology, including mutagenesis, purification optimisation, activity
assay development and tuning of standard structural biology methods for high
temperature analysis. Chapter two is completely related to in silico methods
for rapid analysis and acquisition of NMR relaxation dispersion data. This
involved software development with collaborators of other NMR departments in
Europe and a need for deep theoretical insight in the mathematical equations
of NMR.
This complete separation of domain knowledge may also be an explanation
to why I had so much trouble finishing in time. I owe a lot of thanks to a
multitude amount of people, who generously spent their time helping me solve
all kinds of problems.
It has been a great pleasure to supervise and work together with Casper
de Lichtenberg on the esterase project. Thank you for all your effort and your
independent research, which significally contributed to the project. I wish to
thank my PhD colleague Daniel Stiefler-Jensen, for the countless hours we have
spent together, getting the lab protocols to work under elevated temperature.
It has been a fruitful collaboration, where we were able to strenghten the
development of the project with each of our respective domain knowledge. I am
in depth to Johan Olsen, for help with all crystallisation experiments, booking
and data acquisition at the MAX-Lab synchrotron and solving of the crystal
structure.
I am deply in depth to Edward d’Auvergne who patiently developed my
competencieswithinprofessionalsoftware development. The hundredsof emails
he patiently answered in the slow development of relax into the most efficient
free NMR software available. I was particular enlighten by visiting Edward,
during my short academic stay in the laboratory of Prof. Christian Griesinger,
Göttingen. Maxim Mayzel has been a tremendous help for solving any issues
with regards to processing non-uniform NMR datasets. The biomolecular NMR
course in Gothenburg in the laboratory of Prof. Vladislav Orekhov was a very
fruitful experience with personal discussions of rapid acquisition of NMR data.
I have been so lucky, to have been affiliated with SBiNLab since the start
ii
of my master thesis in 2010. For all those years, I am extremely grateful for
all the advice, discussions, planning, calculations, plotting and writing support
that my supervisor Kaare Teilum has offered. Thank you!
Finally, I wish to thanks all the co-workers of SBiNLab. You make it the
best place to work, think, discuss, grow and smile during all the hours we spent
theretogether. MyfinalgratitudegoestomydearwifeClarissa, whohavebeen
my shining star. Thank you for your patience.
Troels Schwarz-Linnet
Copenhagen, December 2017
iii
Abstract
This thesis is divided into two chapters. The first chapter concerns
the continued work on detection and biophysical characterisation of
post-translational modifications of an archaea esterase from Sulfolobus
islandicus. Thesecondchapterconcernsdevelopmentofinsilico methods
for rapid analysis and acquisition of NMR relaxation dispersion data.
Archaea esterase EstA from Sulfolobus islandicus
This work is a continuation from previous work in the group of Asso-
ciate Professor Qunxin She at Danish Archaea Centre - University of
Copenhagen.
The project is a comparative study of a thermophile-expressed and
mesophile-expressed Sulfolobus islandicus esterase EstA. A considerable
amount of effort has focused on eliminating some minor discrepancies in
constructsandestablishapurificationprotocolforE.coli expressedEstA,
with equal solubility and dimer formation properties as the Sulfolobus
islandicus expression. We also found it necessary to refine the previous
described protocol for enzyme activity assays, since there exists a range
of chemical challenges under elevated temperatures.
We did indeed find the thermophile-expressed EstA being a target
for 1-3x lysine methylation post-translational modifications. We also
confirm that the enzyme Archaeal Lysine Methyl Transferase (aKMT)
is the responsible for this distribution of methylation of lysines. Via
isoelectric focusing gels, we also established that Sulfolobus islandicus
expressedproteinscontainssubpopulationsofmoreacidicspecies. During
the writing of this thesis, we found early literature describing these
subpopulations as being a product of deamidation of asparagine residues.
Allmassspectromicdataacquiredunderthisprojecthasbeenre-examined
during writing of this thesis. Mass determination of previous unknown
peptides from an endoproteinase AspN digestion of Sulfolobus islandicus
EstA, can in some examples be assigned to peptides where an asparagine
has been deamidated to aspartic acid. The in silico AspN digestion map
of EstA, needs to allow cleavage of peptide bonds N-terminal to aspartic
acid residues and asparagine to confirm these masses.
Ourcomparisonbetweenthermophile-expressedandmesophile-expressed
EstA only shows minor differences. The differences between the EstA
construct is most notable, when the enzyme has been exposed to stress-
conditions as elevated temperatures in detergent solution. These minor
differences resemble results presented in other literature. An effort has
beengiventopresentandreviewliteraturehypothesesfortheexplanation
of lysine methylations and compare the methods used in the different
articles.
iv
Rapid analysis and acquisition of NMR relaxation disper-
sion data
Performing a complete NMR relaxation dispersion analysis can be a
challenging process. We engaged in a collaborative software development
with other NMR departments in Europe, to further expand and optimise
the NMR software relax. All mathematical operations in relax was
carefully optimised to work in 5-dimensional data structures, which
increased the calculation speed of relax by a factor 20-40x times.
We review how the software MddNMR efficiently can reconstruct
sparsely recorded NMR relaxation dispersion data. We find that such
datasets can be recorded at 1/4 of normal acquisition time without
comprising the quality. The quality of the reconstruction is performed
on both NMR peak intensity, as well as complete relaxation dispersion
analysis. Only few articles within the field of non-uniform sampling
expands the analysis further than peak intensities. This work can as
such be used as a resource of comparison for the future development of
methods in non-uniform sampling. The ability to reconstruct the NMR
data depends on quality of the sample. We propose a method to assess
the safe non-uniform sampling level, by sampling a fully recorded HSQC
spectrum at different sparsity levels.
All data from the non-uniform sampling project was calculated in
relax to ensure the highest quality of NMR dispersion analysis.
v
Resumé
Denne afhandling er inddelt i to kapitler. Det første kapitel er en
fortsættelseaftidligerearbejdefordetekteringogbiofysiskkarakterisering
afefter-translatereredemodificeringerafenarkæeresterasefraSulfolobus
islandicus. Det andet kapitel omhandler udvikling af digitale metoder for
hurtig analyse og optagelse af NMR dæmpning dispersions data.
Arkæer esterase EstA fra Sulfolobus Islandicus
Arbejdet er en fortsættelse af tidligere udførte undersøgelser i Lektor
Qunxin She’s laboratorie på det danske arkæer center ved Københavns
Universitet.
Projektet er et komparativt studie af en termofil-udtrykt og mesofil-
udtrykt Sulfolobus islandicus esterase EstA. En væsentlig del af arbejdet
harfokuseretpåatfjernemindreforskelleiproteinkonstrukterogetablere
oprensnings protokoller for E. coli udtrykt EstA, med samme opløsnings
og dimer formerings egenskaber som udtrykt i Sulfolobus islandicus. Vi
fandt det også nødvendigt at raffinere de tidligere beskrevne protokoller
for enzym aktivitet, siden der er en række kemiske udfordringer ved høje
temperaturer.
Vi fandt at det termofil-udtrykte EstA protein var metyleret 1-3x
gange på lysiner. Vi bekræftede også at arkæer enzymet ”Archaeal Ly-
sine Methyl Transferase” (aKMT) var ophav til denne distribuering af
metylerede lysiner. Ved hjælp af isoelektriske fokuserings geler, fandt vi
at Sulfolobus islandicus udtrykt protein har mindre populationer af mere
syre karakter. Under skrivningen af denne afhandling, har vi afdækket
tidligere litteratur der beskriver disse populationer som værende et pro-
duktafdeamideringafasparagintilaspartat.Allemassespektrometriske
data i dette projekt er blevet gennemgået på ny under skrivningen af
denne afhandling. Masse bestemmelse af tidligere ukendte peptider fra
en proteinase AspN skæring af Sulfolobus islandicus EstA, kan i nogle
tilfælde blive attribueret til peptider hvor en asparagin er konverteret
til aspartat. Digitale AspN skæringskort af EstA, skal tillade skæring af
N-terminale peptid bindinger efter aspartat og asparagin for at bekræfte
disse masser.
I vores sammenligning mellem termofil-udtrykt og mesofil-udtrykt
EstA fandt vi kun små biofysiske forskelle. Forskellen mellem EstA kon-
strukterne var tydeligst når enzymerne havde været udsat for stress
betingelsersomforhøjettemperaturidetergentopløsninger.Dissemindre
forskelle er også beskrevet i forsknings litteraturen. Afhandlingen inde-
holder en præsentation af de forskellige hypoteser i litteraturen der har
været genstand for diskussion af metyleringens funktion og afhandlingen
sammenholder de forskellige biofysiske metoder der har været anvendt.
vi
HurtiganalyseogoptagelseafNMRdæmpningdispersions
data
At udføre en fuldstændig NMR dæmpnings dispersions analyse kan
være en krævende proces. Vi deltog i et software samarbejde med andre
NMR afdelinger i Europa, for at udvikle og forbedre NMR software
programmet relax. Alle matematiske operationer i relax blev omhyggeligt
optimeret til at kunne håndtere de 5-dimensionelle datastrukturer på
effektivvis.Denneoptimeringgavenforbedringafregnehastighedenmed
en 20-40x faktor.
Vi viser hvordan programmet MddNMR effektivt kan rekonstruere
partielt optagede NMR dæmpnings dispersions data. Vi finder at så-
danne dataset kan optages på 1/4 af den normale optagetid uden at
kompromittere kvaliteten af data. Bestemmelsen af kvaliteten for den
rekonstruerede data er bestemt ved både at sammenligne NMR punkt
intensiteter samt en komplet dæmpnings dispersions analyse. Kun få ar-
tikler indenfor feltet af ikke-uniform optagelsesteknikker analyserer andet
en intensitets sammenligninger. Dette arbejde kan således anvendes som
ressourcetilsammenligningunderfremtidigeudviklingerafikke-uniforme
optagelsesteknikker. Evnen til at rekonstruere NMR data afhænger af
prøvens kvalitet. Vi foreslår en metode til at finde en sikker skæringslinje
for minimum optagetid, ved at digitalt at rekonstruere et fuldt optaget
HSQC spektrum under forskellige virtuelle partielt optagetider.
Alle data fra dette projekt blev analyseret i relax for at sikre den
højeste mulige kvalitet af NMR dæmpnings dispersions analyserne.
Contents
Abstract iii
Resumé v
List of publications ix
1 Post-translational modifications of an archaea esterase 1
1.1 Continuation from previous work . . . . . . . . . . . . . . . . . 1
1.2 Sulfolobus islandicus . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Sulfolobus cytoplasma . . . . . . . . . . . . . . . . . . . 3
1.2.2 Sulfolobus morphology . . . . . . . . . . . . . . . . . . . 4
1.2.3 Sulfolobus metabolism and cell structure . . . . . . . . 6
1.3 Thermophilic enzyme adaptations . . . . . . . . . . . . . . . . 9
1.3.1 Thermal inactivation via conformational scrambling and
deamidation . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.2 Intermolecular interactions that initiate protein aggregation 13
1.4 Examples of prevalent lysine methylations in Sulfolobus . . . . 15
1.4.1 Hypothesis: Is lysine methylation part of a cumulative
effect of intramolecular hydrophobic interactions? . . . . 15
1.4.2 Hypothesis: Is lysine methylation a response to heat-shock? 15
1.4.3 Null Hypothesis: Unmethylated recombinant enzymes is
equally thermostable as the wild-type protein . . . . . . 15
1.4.4 Hypothesis: Unmethylated recombinant enzymes is more
prone to denaturation and aggregation events compared
to the wild-type protein . . . . . . . . . . . . . . . . . . 16
1.4.5 Comment to examples of lysine methylations . . . . . . 19
1.5 Lysine methylation in cell regulation . . . . . . . . . . . . . . . 22
1.5.1 Archaeal Lysine Methyl Transferase - aKMT . . . . . . 24
1.6 EstA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.6.1 Esterase structures . . . . . . . . . . . . . . . . . . . . . 30
1.6.2 Structure of EstA . . . . . . . . . . . . . . . . . . . . . 31
vii
viii CONTENTS
1.6.3 Catalytic triad EstA . . . . . . . . . . . . . . . . . . . . 33
1.6.4 Dimer interface of EstA . . . . . . . . . . . . . . . . . . 36
1.6.5 Lysines in EstA . . . . . . . . . . . . . . . . . . . . . . . 37
1.7 ConclusionsfrompaperI+II-Theextraordinarythermalstability
of EstA from S. islandicus is independent of post translational
modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2 RapidanalysisandacquisitionofNMRrelaxationdispersion
data 43
2.1 Motivation for paper III - relax: the analysis of biomolecular
kinetics and thermodynamics using NMR relaxation dispersion
data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1.1 Optimisation of relax libraries for rapid analysis . . . . 45
2.1.2 Effective handling of 5-dimensional NMR data . . . . . 46
2.1.3 Example graphs of dispersion models . . . . . . . . . . . 49
2.2 Comments to paper IV - Non-uniform sampling of NMR relax-
ation data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Bibliography 61
Appendices 73
Materials and methods: EstA . . . . . . . . . . . . . . . . . . . . . . 74
Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Protein expression & Purification . . . . . . . . . . . . . . . . . 78
X-ray crystallography of EstA . . . . . . . . . . . . . . . . . . . 95
aKMT structural alignment of Chu et al. (2012) . . . . . . . . . . . 97
Blast and multiple alignment of EstA . . . . . . . . . . . . . . . . . 99
Trypsin and AspN digestion of SisEstA . . . . . . . . . . . . . . . . 101
In-silico digestion of EstA in mMass . . . . . . . . . . . . . . . . . . 103
DatacollectionandrefinementstatisticsforcrystalstructureofSisEstA
5LK6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Papers 105
PaperI-TheextraordinarythermalstabilityofEstAfromS.islandicus
is independent of post translational modifications . . . . . . . . 105
Paper II - An esterase activity assay suitable for use at temperatures
up to 90C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Paper III - relax: the analysis of biomolecular kinetics and thermody-
namics using NMR relaxation dispersion data . . . . . . . . . . 127
Paper IV - Non-uniform sampling of NMR relaxation data . . . . . . 130