influence of aromatic and negatively charged side chains on membrane protein stability and ...

297 Pages·2016·15.13 MB·English

INFLUENCE OF AROMATIC AND NEGATIVELY CHARGED SIDE
CHAINS ON MEMBRANE PROTEIN STABILITY AND HYSTERESIS

by
Sarah Kempka McDonald

A dissertation submitted to Johns Hopkins University in conformity with the
requirements for the degree of Doctor of Philosophy

Baltimore, Maryland
February 2016

© 2016 Sarah Kempka McDonald
All Rights Reserved
Abstract
Fundamental to the field of biophysics is quantitating and understanding the
physical forces responsible for the interactions of biomolecules. Membrane proteins are
presented with cellular bilayers that provide a unique medium in which hydrophobic
sequences can fold. Knowledge of the energetics of protein-lipid interactions is thus vital
to understand cellular processes involving membrane proteins. In this work, we have
addressed two important questions in biology: how do the aromatic side chains respond
to the water density gradient in the membrane and how can we engineer membrane
proteins to fold reversibly at a neutral pH? To address the first question, we used a host-
guest mutational strategy with an outer membrane protein (OmpLA) to measure the
water-to-lipid side chain transfer free energies as a function of membrane depth. The
experimental measures demonstrated an energetic gradient in Trp and Tyr transfer, which
was most favorable at the membrane interface. Complimenting this study with molecular
dynamics simulations revealed the energetics were correlated with the changing water
density naturally present in the membrane. To address the second question, we used the
OmpLA folding system that is reversible at pH 3.8, but displays large hysteresis in
unfolding/folding denaturant titrations at pH 8. We hypothesized that the ionization states
of negative side chains were responsible for closure of the hysteresis gap. This idea was
tested by introducing AspAsn and GluGln mutations to neutralize the charges in
various regions of the molecule. Although hysteresis could not be completely eliminated
at pH 8, we show that neutralization mutations in the loops and turns were responsible for
a 60% closure in the hysteresis gap relative to the WT protein. Results from these
ii
projects may be applied to model protein structures in the membrane and will aid in the
engineering of membrane proteins.

Thesis advisor: Dr. Karen G. Fleming
Second reader: Dr. Jeffrey Gray
Thesis committee: Dr. Doug Barrick, Dr. Bertrand Garcia-Moreno, & Dr. Albert Lau

iii
Acknowledgements
First I want like to thank my family. I would like to give a special thanks to my
grandmother Gail Herron. Without your love, faith, and family traditions none of us
would be who we are today. In memorium, I would like to thank my grandfather Jack
Herron, who passed away while I was writing my thesis. I wish you would have seen me
graduate, but I know how proud you were of me and I will carry that in my heart. I would
next like to thank my mother Dianne Herron and my father Neil Kempka for allowing me
to follow my dreams and supporting me along the way. I feel as I have the best of both of
your traits; my mother has blessed me with her creativity and my father with his logic.
Both have blessed me with their good looks. I would also like to thank my new parents,
Jaime and Patti McDonald for loving me as your own. I couldn’t be happier with the
family I have chosen. Being over 1000 miles away for 6 years has been a challenge, but
the experience has made me appreciate how special and great all of you are. Thank you
for your love and support throughout the years.
I would next like to thank my wonderful husband Riley McDonald. We have been
through a lot together over the past twelve years. In all of those years I have been in
college, and you have constantly adapted your life to be with me. From driving back-and-
forth to Duluth nearly every weekend, to making the great leap to Baltimore, you were
there. Thank you for your support and understanding. You are so much more than my
husband, you are my best friend and I look forward to the many years and adventures we
have ahead of us.
iv
Thanks to the past and present members of the Fleming lab. It has been really fun
to see the lab grow and change over the years. When I first joined in 2011, Emily Danoff
was the only graduate student. Not only did she train me, she challenged me, and I will
always love her for that. Over the years I have had the pleasure of working with two post-
docs: Nathan Zaccai and Dennis Gessmann. Thank you for bringing your talents into the
lab-you have both taught me so much. Cliff Sandlin joined the lab the year after me, and I
am appreciative of his thoughtful insight on my projects. When Ashlee Plummer joined
the next year, she brought a positive energy that not only motivated me, but also made
coming to work every day a joy. I will miss starting my day with our chats over coffee.
Thanks to Henry Lessen, for laughing at my jokes and whose own humor brightened my
days. I am also grateful for Dagan Marx, who will carry on the stability measurements
after my departure. I also cannot forget all of the bright undergraduates we have had in
our lab: Sam Chirtel, Will Chung, Margo Goodall, Mike Yamakawa, Quenton Bubb, and
Shawn Costello. Finally, I would like to thank Pat Fleming. Thank you Pat for being my
second mentor and reminding me to always look at the chemical structure!
Finally, huge thanks to all members of my thesis committee: Jeff Gray, Doug
Barrick, Bertrand Garcia-Moreno and Albert Lau. Without you challenging my data, it
would not be what it is today. Last, but not least, I would like to thank my thesis advisor
Karen Fleming. Karen, I will never be able to express how grateful I am. You have taught
me invaluable skills not only in science, but in life. Working with you has allowed me to
grow my confidence and develop into a great scientist. Thank you for being so kind and
caring. I am so glad that I was able to spend the last 6 years working with you.

v
List of abbreviations
Agg Aggregation
AIC Akaike’s information criterion
ASA Accessible surface area
ATP Adenosine triphosphate
ATPase Enzymes that hydrolyze ATP
AUC Analytical ultracentrifugation
BAM complex -barrel assembly machine complex in outer membrane
CD Circular dichroism
COM Center of mass
DLPC 1,2-dilauroyl-sn-glycero-3-phosphocholine
DOF Degrees of freedom
DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
DTNB 5,5′-dithio-bis-(2-nitrobenzoic acid)
EDTA Ethylenediaminetetraacetic acid
Exp. Exponential
Gau. Gaussian
GdnHCl Guanidine hydrochloride
GTP Guanosine triphosphate
GTPases Enzymes that hydrolyze GTP
HDS 1-hexadecanosulfonic acid
HEPC 2-hexadecanoyl-1-ethylphosphorylcholine
IF In fusion
IM Inner membrane
IMP Inner membrane protein
IPTG Isopropyl β-D-1-thiogalactopyranoside
Lin. Linear
LPS Lipopolysaccharide
vi
LUV Large unilamellar vesicle
MARCC Maryland advanced research computing center
MD Molecular dynamics
NMR Nuclear magnetic resonance
OM Outer membrane
OMP Outer membrane protein
OmpA Full length OmpA, includes the periplasmic domain
325
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
POPG 1-palmitoyl -2-oleoyl-sn-glycero-3-phosphoglycerol
QC Quickchange
Res. Residuals
RGD Rayleigh-Gans-Debye
RMSD Root mean squared
S.C. Side chain
SB3-14 3-N,N-Dimethylmyristyl-ammonio-propanesulfonate
SDS Sodium dodecyl sulfate
Sig. Sigmoidal
SUV Small unilamellar vesicle
TB Terrific broth
TRIS Tris(hydroxymethyl)aminomethane
uOMP Unfolded outer membrane protein
VMD Visual molecular dynamics

vii
List of proteins
BamA -barrel and POTRA component of BAM complex
BamBCDE Additional proteins in BAM complex
bR Bacteriorhodopsin
DegP Periplasmic chaperone and protease
DGK Diacyl glycerol kinase
FadL Fatty acid transporter in outer membrane of bacteria
FtsY SRP peripheral membrane protein receptor
KdpD Potassium sensor in inner bacterial membrane
OmpA Outer membrane protein A, phage receptor
OmpC Outer membrane protein C, osmoporin
OmpLA Outer membrane phospholipase A
OmpT Outer membrane protein T
OmpW Outer membrane protein W
OmpX Outer membrane protein X
PagP Enzyme in outer membrane, transfers a palmitate chain from a
phospholipid to lipid A
POTRA P1-P5 Polypeptide translocation associated domains in BAM complex
SecA ATPase that provides the energy for translocation through
SecYEG
SecB Cytosolic chaperone for proteins that pass through the inner
membrane
SecYEG Protein translocation channel in bacteria
Skp Seventeen kiloDalton protein, periplasmic chaperone
SRP Signal recognition particle, directs polypeptide chain to
translocon
SurA Periplasmic chaperone and prolyl isomerase
TF Trigger factor, ribosome binding chaperone
Wza Outer membrane lipoprotein Wza, polysaccharide translocon

viii
Table of contents
Abstract ii
Acknowledgements iv
List of abbreviations vi
List of proteins viii
Table of contents ix
List of tables xi
List of figures xv

Chapter 1 Introduction 1
1.1 Overview and perspectives 1
1.2 Structures of membrane proteins and lipid bilayers 2
1.3 Biogenesis of membrane proteins in gram negative bacteria 6
1.4 Membrane protein stability measurements in vitro 13
1.5 The role of aromatic side chains in membrane protein stability 24
and folding
1.6 Overview of thesis 35
1.7 Figures 41

Chapter 2 Aromatic side chain water-to-lipid transfer free energies in 50
membrane proteins reflect the water density gradient across
the bilayer normal
2.1 Abstract 50
2.2 Introduction 51
2.3 Materials and methods 55
2.4 Results 65
2.5 Discussion 77
2.6 Tables and figures 82

ix
Chapter 3 Molecular dynamics simulations of aromatic side chain 133
variants show site specific behavior in response to the
membrane and protein environment
3.1 Abstract 133
3.2 Introduction 134
3.3 Materials and methods 137
3.4 Results 141
3.5 Discussion 148
3.6 Tables and figures 151

Chapter 4 Neutralization of negative charges in the loops and turns 189
partially closes the hysteresis gap in outer membrane
protein folding
4.1 Abstract 189
4.2 Introduction 190
4.3 Materials and methods 194
4.4 Results 201
4.5 Discussion 212
4.4 Tables and figures 218

Chapter 5 Concluding remarks 246

References 254

Curriculum vitae 278

x