KJE-3900 Master's Thesis in Chemistry Flexible membrane active antimicrobial tripeptides with ...

105 Pages·2010·13.06 MB·English

KJE-3900
Master’s Thesis in Chemistry
Flexible membrane active antimicrobial
tripeptides with stability towards chymotryptic
degradation
Geir Isaksen
May, 2010
Faculty of Science
Department of Chemistry
University of Tromsø

KJE-3900
Master’s Thesis in Chemistry
Flexible membrane active antimicrobial
tripeptides with stability towards chymotryptic
degradation
Geir Isaksen
May, 2010
Faculty of Science
Department of Chemistry
University of Tromsø

Flexible membrane active antimicrobial
tripeptides with stability towards
chymotryptic degradation
Geir Isaksen
May, 2010
Keywords: Antimicrobialpeptides;tripeptides;geometryoptimization;molec-
ulardynamics; phasespace; faceflipping; membraneinteraction; chymotrypsin;
docking; isothermal titration calorimetry; molecular modelling; crystallization;
crystal structure.
Abstract: Inrecentyearsantimicrobialpeptideshavegainedalotofattention
due to their potential as a new generation of antibiotics combating the growing
problemofantibioticresistance. Itisbelievedthattheamphipathicstructureof
cationicpeptidesisakeyfeatureforantimicrobialactivity,andthatthisenables
them to interact with the bacterial cell membrane. The conformational space
of a range of cationic tripeptides have in this project been studied in solvent
usingdensityfunctionaltheoryandmoleculardynamicssimulations. Theresults
indicate that the cationic tripeptides are able to change between different, but
equally stable, conformations that are both amphipathic and non-amphipathic,
a property referred to as face flipping. Based on this, face flipping is proposed
to be a key feature for the membrane interaction mechanism. The tripeptides
mode of interaction was therefore studied with cellular model systems in more
detail using MD simulations. The results show that the peptides first interact
with the negatively charged head groups of the membrane with their cationic
charges and then flip the hydrophobic groups into the membrane bilayer. The
results thus provide strong support to the face flipping hypothesis.
A problem with antimicrobial peptides is that oral administration is diffi-
cult due to the degradation by digestive enzymes. The stability towards chy-
motryptic degradation has therefore been investigated by probing the S1, S1’
and S2’ binding pockets with unnatural amino acid side chains. The effect
of different side chain substitutions were examined by combining isothermal
titration calorimetry, crystallization experiments and extensive molecular mod-
elling. Through these studies it was possible to investigate the preferential
binding of several relevant unnatural amino acid side chains to the subsites of
chymotrypsin. Important structural and mechanistic features were revealed in
a fashion not feasible through the use of native peptide substrates. It was also
found that proteolytic stability can be controlled not only by probing the S1
pocket, but also the notably less studied S1’ pocket.
1
2
Acknowledgments
ThepresentedworkwasdoneattheUniversityofTromsø intheperiodbetween
August 2008 and May 2010. I would like to greatly acknowledge my supervi-
sor Bjørn Olav Brandsdal for giving me the opportunity to be a part of a very
interesting and challenging project, and for always supporting and guiding my
ideas in the right direction. Thank you for all the necessary help and feedback
through the writing process, and for giving me the freedom to grow, not only
as a chemist, but also as a scientist. I would like to bring a special thanks to
Hanna-Kirsti Leiros who has been of great help during the crystallization part
oftheproject;Iappreciatethetimeyouspentexplainingandsharingfromyour
experience as a crystallographer making the world of structure determination a
lot easier to maneuver in. I would also like to thank Ronny Helland for all the
nice discussions we have had during my master, for all the advises concerning
crystallization conditions, and for donating chymotrypsin. Johan Svenson and
Rasmus Karstad for contributing with Half-life assays and MIC values. Johan
for always bringing me more peptides for my never ending crystallization ex-
periments. Kathrin Helen Hopman for all her computational help and personal
effort during my attempt to geometry optimize the petides in solvent with den-
sity functional theory. Annfrid Sivertsen for always offering a helping hand and
for borrowing me peptides when I was all out (and Johan could not bring me
more fast enough). Thank you for all the time you spent teaching me with the
ITC and introducing me to the world in the “wet lab”. I would also like to
thankBjarteA.Lundforlearningmehowtouse thecrystallizationrobot(sev-
eral times). Antoine Durdek for his mathematical expertise and for making our
office a nice and funny place to be in. Arnfinn Hykkerud Steindal for providing
a helping hand whenever I was struggling with linux or LATEX.
As a member of both NorStruct and CTCC I have met incredibly many
friendly and helpfull people. I would therefore like to thank all the present and
formermembersofNorStructandtheCTCCthathavecontributedtomywork
and working environment. All my friends for skiing, serious squash playing,
concerts(playingandlistening), coldbeersatflyt, andingeneralforreminding
me that there is a life outside the office during the hard writing period of this
thesis. Iwouldalsoliketothankmyparentsforalltheirunconditionalsupport.
My special heartfelt gratitude goes to my fiancee, Silje Jørgensen, for all
her love and support, and for having a huge tolerance for my ever workaholic
tendencies. Thank you for making all of this possible ♥.
Geir Isaksen
3
4
Abbreviations & symbols
∇2 Laplace operator; ∇2 = ∂2 + ∂2 + ∂2
∂x2 ∂y2 ∂z2
χ Atomic orbital
φ Molecular orbital
Φ Slater determinant
Ψ Many electron wave function
AO Atomic orbital
Agp L-2-amino-(3-guanidino)propanoic acid
Amphiphile Chemical compound possessing both hydrophilic and
lipophilic properties. (such compunds are called
amphiphilic or amphipathic)
AMPs Antimicrobial peptides
App L-2-amino-3-(4-aminophenyl)propanoic acid
ATTC American type culture collection
Bip L-biphenyl alanine
Bn Benzyl
BPTI Bovine Pancreatic Trypsin Inhibitor
CAP Cationic Antimicrobial Peptide
CC Coupled cluster
CHT Chymotrypsin
CI Configurational interaction
CM Cytoplasmic membrane
DFT Density functional theory
DIIS Direct Inversion of the Iterative Subspace
(convergence algorithm)
DI Differential algorithm
Dip L-β-phenyl-phenylalanine
DPG Diphosphatidylglycerol
DPI Cruickshank’s dispersion precision index for coordinate error:
DPI =(cid:112)(Natom/(Nrefl−Nparam))RfactDmaxcompl−31
EC Energy correlation
Et Ethyl
FISA Hydrophilic solvent accessible surface area
Force field A sum of different molecular mechanics terms expressing the
energy of a system
FOSA Hydrophobic solvent accessible surface are
GA Genetic algorithm
5
ABBREVIATIONS & SYMBOLS
GGA Generalized gradient approximaton
Gpp L-2-amino-3-(4-guanidinophenyl)propanoic acid
GRP Generalized random phase
Har L-2-amino-(4-guanidino)hexanoic acid
HF Hartree-Fock
ITC Isothermal titration calorimetry
LSDA Local spin density approximation
MBPT Many-body perturbation theory
MC Monte carlo
MD Molecular dynamics
MIC Minimal inhibitory concentration
MR Molecular replacement
MRSA Methicillin-resistant staphylococcus aurerus
MO Molecular orbital
OM Outer membrane
Orn L-ornithine
PCM Polarizable continuum model
PE Phosphatidylethanolamine
Ph Phenyl
Phatogen Organism, usually microorganism, that causes disease
PISA π component of the solvent accessible surface area
POPE 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanol-amine
PG Phosphatidylgylcerol
PRCG Polak-Ribiere-type conjugate gradient
QM Quantum mechanics
RBD Rigid-body docking
RMSD Root mean square distance between pairs of atoms
(cid:113)
RMSD= PNiN=aa1ttoommssd2i; di =|xi−x(cid:48)i|
RP-HPLC Reversed Phase High Performance Liquid Chromatography
SA Simulated annealing
SASA Solvent accessible surface area
SCF Self-Consistent Field
SCRF Self-consistent reaction field
Tbt β-(2,5,7-tri-tert-butylindol-3-yl)alanine
UFF Universal Force Field
VRE vancomycin-resistant Enterococcus faecium
XP Extra precision (in Glide docking)
6