Boston UniversityOpenBU http://open.bu.eduTheses & Dissertations Boston University Theses & Dissertations

2015

B9-17: A suitable construct forapolipoprotein B-containinglipoprotein assembly studies

https://hdl.handle.net/2144/13724Boston University

BOSTON UNIVERSITY

SCHOOL OF MEDICINE

Thesis

B9-17: A SUITABLE CONSTRUCT FOR APOLIPOPROTEIN

B-CONTAINING LIPOPROTEIN ASSEMBLY STUDIES

by

MELYORISE SEPULVEDA CHERVONY

B.Sc., University of Puerto Rico, 2010

Submitted in partial fulfillment of the

requirements for the degree of

Master of Arts

2015

c© 2015 byMELYORISE SEPULVEDA CHERVONYAll rights reserved

Approved by

First Reader

C. James McKnight, PhDAssociate Professor of Physiology and Biophysics

Second Reader

Esther Bullitt, PhDAssociate Professor of Physiology and Biophysics

ACKNOWLEDGEMENTS

Fabiola Gianotti, particle physicist and future Director-General at CERN, once said: “This

job is a great scientific adventure. But it’s also a great human adventure.” Just like quantic

entities, to me, this quote has duality. I have spent the past few years going through a

great scientific adventure. I have learned an inmensurable amount of scientific information:

principles, theories, models, but I also have learned about the human aspect of science.

My human adventure has been two-fold. Learning all this and being part of the Graduate

Medical Sciences, have helped me see the clinical part of research in a different light. I was

able to see how one little advance in the field could mean a healthier life for a person. More

than personal motivation, the knowledge that my findings could help people suffering from

heart disease, gave me a sort of boost. Specially to finish this Masters thesis. I have also

interacted with a lot of humans involved in the quest for knowledge and that has brought

the second aspect of my human adventure. I would like to take the opportunity to highlight

those humans that have been part of this adventure. Without them my experience would

have been one-dimensional and we all know how boring that can be.

Foremost, I would like to express my sincere gratitude to my advisor Dr. C. James

McKnight for the continuous support of my Masters study and research, for his patience,

motivation, enthusiasm and knowledge. His guidance helped me in all the time of research

and writing of this thesis.

Besides my advisor, I would like to thank: Dr. Esther Bullitt, Dr. Olga Gursky, and

Dr. David Atkinson, for their encouragement, guidance, insightful comments and hard

questions.

I thank my fellow lab mates, Jacob Turnbull and Humra Athar for the stimulating

discussions, and for all the fun we had in the lab. To my friends, colleagues, faculty and

staff in the Biophysics department. In particular, I am extremely grateful for unconditional

support of Madhurima Das, Deborah Heller, Andrew Bogorad and Nour Alsabeeh. For the

many hours of help they selflessly gave me, for their positive and constructive feedback and

iv

for all the great moments we spent together while working to do our part in the research

world. Also I would like to thank my friends at the University of Puerto Rico. In particular,

I am grateful to Dr. Gustavo E. Lopez for enlightening me with the first glance of research.

I wouldn’t have been able to do and complete this without the support of my loved

ones. I would like to thank my parents, Melvin and Yoly, for giving me the means and

opportunities I’ve had since birth. There is no way to repay you for your patience and

support. To Julian, my companion, friend and partner, thanks for helping put the pieces

together at this final stage and for keeping me balanced. And lastly, I would like to thank

my friends for believing in me and making my life whole. Diane, Phillip and Marıa, Thanks.

I would love to mention all the people that have in one way of another been part of this

journey, but I think that might take up more than half of the contents of this work. The

lack of itemization does not lessen in any way the impact of any of them. I hold each and

everyone dear to my heart and I thank them all for the fraction of life we share.

v

B9-17: A SUITABLE CONSTRUCT FOR APOLIPOPROTEIN

B-CONTAINING LIPOPROTEIN ASSEMBLY STUDIES

MELYORISE SEPULVEDA CHERVONY

ABSTRACT

Atherosclerosis, hardening and narrowing of the arteries, is the principal underlying

cause of heart attacks, strokes, and peripheral vascular disease, which kills more than

600,000 Americans each year. High plasma levels of low-density lipoproteins (LDL) are

linked to the formation of atherosclerotic plaques in arteries. LDL is the last metabolic

product of very low-density lipoprotein (VLDL), which is secreted from the liver along with

one molecule of apolipoprotein B (apoB). Current therapies to control levels of LDL include:

cholesterol synthesis inhibitors or statins, low-fat diets and antisense oligonucleotides to

reduce cholesterol levels. Recent studies recommend lower clinical levels of plasma LDL to

maintain an individual’s health, especially of those who have already developed atheroscle-

rotic plaques. However, existing therapies are often unable to achieve these aggressive

limits. Furthermore, patients have shown various levels of intolerance to these treatments.

In order to develop new, targeted drugs, that can control LDL levels with minimal side

effects, it is imperative to understand, in detail, the process of apoB-containing lipoprotein

formation.

ApoB is one of the largest human proteins known (4563 residues) and previous attempts

to solve the structure have been unsuccessful, mainly due to analyzing the protein as a whole

or by large sections. To advance the field we will go by a different approach. I present here

a construct that represents roughly 8% of the whole protein, apoB9-17 (residues 430 to

782). This section of the protein is believed to play a pivotal role in the assembly process of

LDL. My hypothesis is that this construct will be well-behaved and suitable for structural

and functional analysis. The study shows that apoB9-17 can be produced in considerable

quantities from bacterial cells and can be purified by means of a 6-histidine tag with a good

yield. Furthermore, circular dichroism analysis shows the construct contains the expected

vi

secondary structure at room temperature and is stable at a wide temperature range (50 to

70 ◦C) at low concentrations.

The construct here described will be useful to test the effect of mutations such as

the one found in patients with Familial hypobetalipoproteinemia (FHBL). Furthermore,

this construct contains two regions believed to be of vital importance for LDL particle

formation: the alpha-helical region (residues 430 to 570) is believed to associate with MTP

at the initial stages of LDL formation and the c-sheet (residues 614 to 782), which may form

part of the lipid recruiting process. Both essential aspects to ultimately develop therapies

that can modulate VLDL particle formation.

vii

TABLE OF CONTENTS

Title i

Copyright Page ii

Reader Approval Page iii

Abstract vi

Table of Contents ix

List of Figures x

1 Introduction 1

2 Materials and Methods 18

2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1.1 Laboratory Equipment, Chemicals and Kits . . . . . . . . . . . . . . 18

2.1.2 Cells and Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1.3 Solutions and Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1.4 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2.1 Construct Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2.2 Construct Purification . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.2.3 Construct Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.2.4 Size Exclusion Chromatograpy . . . . . . . . . . . . . . . . . . . . . 23

viii

3 Results 24

3.1 Expression and Purification of ApoB9-17 . . . . . . . . . . . . . . . . . . . . 24

3.2 Spectral Properties of ApoB9 - 17 . . . . . . . . . . . . . . . . . . . . . . . 30

3.3 Aggregation State of ApoB9-17 . . . . . . . . . . . . . . . . . . . . . . . . . 33

4 Conclusions 35

Bibliography 38

Curriculum Vitae 44

ix

LIST OF FIGURES

1.1 Lipoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Cholesterol-Rich Lipoprotein Metabolism . . . . . . . . . . . . . . . . . . . 6

1.3 Low-Density Lipoprotein Model . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.4 ApoB Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.5 Lipoprotein Secretion and Assembly Model . . . . . . . . . . . . . . . . . . 12

1.6 LDL Particle Electron Microscope Reconstructions . . . . . . . . . . . . . . 13

1.7 Lipovitellin N-terminal Homology Model of ApoB20 . . . . . . . . . . . . . 14

1.8 ApoB20 Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1.9 ApoB9-17 Model Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.1 SDS-PAGE Analysis of Expressed ApoB9-17 - Before Sonication . . . . . . 25

3.2 SDS-PAGE Analysis of Expressed ApoB9-17 - After Sonication . . . . . . . 26

3.3 Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.4 SDS-PAGE of ApoB9-17 - Purification . . . . . . . . . . . . . . . . . . . . . 29

3.5 CD Spectra of ApoB9-17 - Scan . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.6 CD Spectra of ApoB9-17 - Thermal Denaturation . . . . . . . . . . . . . . . 32

3.7 FPLC - Size-Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . 34

x

LIST OF SYMBOLS AND ABBREVIATIONS

ApoB9-17 . . . . . . . . . . . . . . . . . . . . . . . . Section 9% to 17% of Apolipoprotein B

ApoB48 . . . . . . . . . . . . . . . . . . . . . . . . . . Section 0% to 48% of Apolipoprotein B

ApoB100 . . . . . . . . . . . . . . . . . . . . . . . . . Denotes the full length of Apolipoprotein B

BL21(DE3) . . . . . . . . . . . . . . . . . . . . . . . Chemically Competent E. coli Cells Suitable

for Transformation and Protein Expression

BME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-Mercaptoethanol

CD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circular Dichroism

DGAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diglyceride Acyltransferase

ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endoplasmic Reticulum

FHBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . Familial Hypobetalipoproteinemia

IPTG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isopropyl β-D-1-thiogalactopyranosid

LDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low-Density Lipoprotein

LLTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Large Lipid Transfer Protein

LPL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipoprotein Lipase

mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . Messenger Ribonucleic Acid

MTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microsomal Triglyceride Transfer Protein

OD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optical Density

SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis

xi

Introduction

Atherosclerosis is the principal underlying cause of heart disease. It is the number one cause

of death of both men and women in the United States, killing over 600,000 Americans each

year [CDC, 2013]. Atherosclerosis is characterized by the formation of plaques inside the

arterial walls, narrowing the space and thus restricting the blood flow. Plaques can also

rupture, creating a blood clot that can result in a heart attack or stroke. Atherosclerosis

is a very complex disease and there are many factors that are believed to play a role in

its occurrence. The most prominent of these factors is the presence of high levels of low-

density lipoproteins (LDL).

Lipoproteins

Lipoproteins are lipid transport particles. The human body needs fat for things like

energy and vitamin absorption. Fat is water-insoluble and the body is roughly 80% water.

Although a small amount of fat can travel through the body without any help, it does not

meet the needs of the organism. Here is where lipoproteins are useful. Lipoproteins have

a structure that provides an amphipathic shell, ideal for lipid transport (Figure 1.1) [Berg

et al., 2010].

Lipoproteins have a monolayer of phospholipids, as seen in Figure 1.1. The polar heads

of the phospholipids face the outer part of the lipoprotein, which is compatible with the

mainly aqueous composition of the human body. Conversely, the apolar tail faces the in-

ner part of the particle which is rich in lipids, such as cholesterol esters and triglycerides.

Additionally, there is a small amount of cholesterol found in this phospholipid monolayer.

Lastly, lipoproteins are characterized by proteins embedded in the phospholipid layer, called

1

apolipoproteins. Apolipoproteins have amphipathic alpha-helices as a common structural

motif. Apolipoprotein B, the subject of this thesis, is unique in that it has large amounts

of amphipathic β structure in addition to amphipathic helices. These amphipathic protein

structures allow the protein to interact with the core, but yet ’float’ on the surface of the

lipoprotein particle to carry out its function.

There are different types of lipoproteins. They differ from each other by the ratio

of proteins to lipids, and in the apolipoproteins and types of lipids that they contain.

Lipoproteins are classified by their density [Berg et al., 2010]:

• Chylomicrons (CM) - Primarily transport triglycerides from the intestines to the

liver, skeletal muscle, and to adipose tissue.

• Very low density lipoproteins (VLDL) - Transport (newly synthesized) triacyl-

glycerol from the liver to adipose tissue.

• Intermediate density lipoproteins (IDL) - Are derived from VLDL and are

intermediate in size and density between VLDL and LDL.

• Low density lipoproteins (LDL) - Transport cholesterol from the liver to cells of

the body. LDL is also known as ”bad cholesterol.”

• High density lipoproteins (HDL) - Work inversely from all other lipoproteins.

They collect cholesterol from the body’s tissues, and bring it back to the liver. HDL

is also known as ”good cholesterol.”

Apolipoproteins

Apolipoproteins associate with lipoproteins and constitute a structural component.

Their presence is pivotal to lipoprotein function. Apolipoproteins are mainly responsi-

ble for the transport of lipoproteins. Additionally, they are involved in the regulation of

2

lipoprotein metabolism by acting as lipid transfer carriers, enzyme cofactors and recep-

tor ligands. Thus, the apolipoprotein associated with a given lipoprotein determines its

structure, metabolism and interactions.

Apolipoproteins can be classified in under two major types: the exchangeable (apoA,

apoC, apoE) and the non-exchangeable (apoB) [Pownall and Gotto, 1992]. Exchangeable

lipoproteins are small and soluble. They can exchange between lipoprotein classes and

acquire lipids while circulating. Non-exchangeable apolipoproteins are large and water

insoluble [Segrest et al., 1994a]. ApoB100 and apoB48 are the two, naturally-occurring,

non-exchangeable apolipoproteins in the human body. They form part of VLDL and stay

with the particle throughout its various metabolic changes until its removal via LDL re-

ceptor in the liver. The structure of apoB is believed to be the main reason for this

characteristic.

3

Figure 1.1: Cartoon Representation of a Lipoprotein Particle: The structure of

a lipoprotein is ideal for their function. Lipoproteins have a neutral lipid core, primar-

ily triglycerides and cholesterol esters. Encapsulating that core, there is a monolayer of

phopholipids and cholesterol. Their amphipathic character of this monolayer shields the

core from the aqueous environment. Lastly, we find the apolipoproteins, which are charac-

teristic of each lipoprotein. Also characteristic of each lipoprotein, is the weight percentage

of each one of its components.

4

Low-Density Lipoproteins and the Lipid Metabolism

LDL, also known as “bad cholesterol“, is a complex particle high in cholesterol esters.

LDL is the metabolic product from a much less dense particle called very low-density

lipoprotein (VLDL). Both form part of the essential biological process of lipid metabolism

in the human body. Figure 1.2 outlines the major components and steps of a subset of

the lipid metabolism; the cholesterol-rich lipoproteins. The metabolism of these lipopro-

teins can be divided into the endogenous pathway (blue arrows) and the exogenous pathway

(green arrows) [Daniels et al., 2009]. The endogenous pathway includes lipoproteins synthe-

sized by the liver: VLDL, IDL and LDL. They transport endogenous triglycerides (TG) and

cholesterol throughout the body. 1) VLDL originates from the liver as a triglyceride-rich

particle. Associated with each VLDL particle is one molecule of apolipoprotein apoB100

(the entire sequence of apoB). 2) Once in the blood stream, it interacts with lipoprotein

lipase (LPL) which hydrolyzes TGs from the particle core to fatty acids and transport them

to the peripheral tissues. 3) The concentration of triglycerides diminishes and cholesterol

and cholesterol esters are added until the triglycerides concentration is less than cholesterol

esters forming intermediate-density lipoprotein (IDL), a cholesterol-rich particle. 4) Hep-

atic lipase (HTGL) then metabolizes IDL into LDL, making LDL the last metabolic step

in this pathway. ApoB100 maintains its association with all the metabolic products of the

original VLDL particle. A single partial copy of apoB is also naturally present as apoB48

(first 48 % of the sequence of the protein), associated with chylomicrons that are metabo-

lized in a manner similar to VLDL to produce chylomicron remnants. The remnants are

cholesterol-rich particles. VLDL, IDL and chylomicrons associate with other lipoproteins

and other apolipoproteins as well (not shown) to carry fats throughout the body.

5

Figure 1.2: Cholesterol-Rich Lipoproteins Metabolism: ApoB is an essential com-

ponent of the lipid metabolism pathway. Exogenous pathway is indicated by the green

arrows. Endogenous pathway is indicated by the blue arrows. Steps 1 through 4 are de-

scribed in the text.

Image adapted from: [Donelly, 2012], [Ellis-Christensen, 2015] and [Charves, 2008]

6

Low-Density Lipoproteins and Atherosclerotic Plaques

LDL is the smallest, most dense particle of the apoB-containing lipoproteins. LDL

particles have a shell formed by a single molecule of apolipoprotein B-100 (apoB100) and

a monolayer of phospholipids and cholesterol with a core rich in cholesterol esters and

triglycerides (Figure 1.3). Extensive study of the effect of LDL levels in the human body

solidify the theory that LDL initiates the formation of atherosclerotic plaques. One theory

is that LDL penetrates through dysfunctional endothelium cells into the walls of the arter-

ies [Bonetti et al., 2003]. LDL trapped inside the arterial walls triggers a complex chain of

events (including inflammation) that ultimately ends in the formation of an atherosclerotic

plaque.

7

Figure 1.3: LDL Model: LDL particle size varies between 20 to 25 nm. It has a hydropho-

bic lipid core of cholesterylester and triglyceride molecules, which make up more than 40%

of particle mass surrounded by a phospholipid monolayer corresponding to about 20% of

particle mass. One single copy of apo-B100 is embedded in the surface monolayer, partially

penetrating the core and covering about 40-60% of the surface area. The N-terminal end of

apo-B100 (apoB20) is hydrophilic and shows a high homology to lamprey lipovitellin [Seg-

rest et al., 1999].

8

Regulation of LDL uptake to reduce circulating LDL can help decelerate plaque forma-

tion. This task is not simple, as there are multiple factors that affect the process, such as

smoking, high blood pressure and diabetes. The most common therapy for LDL reduction

is the use of statins. Statins inhibit HMG CoA reductase which in turn inhibits the endoge-

nous cholesterol synthesis pathway (known as the acetyl CoA pathway). Depravation of

endogenous cholesterol results in an increase of LDLRs, which lowers plasma LDL [Brown

et al., 1978] by increasing key cell surface receptors that bind to apoB [Anderson et al.,

1977] [Goldstein et al., 1979]. Statins do have the limitation, they cannot successfully

treat patients that already have atherosclerotic plaques present. In addition, a significant

percentage of patients cannot tolerate statin treatment. Therefore the quest for additional

therapies to treat atherosclerosis is not over.

Another way to attack the problem is to target LDL secretion. To do this, we need

to have a better understanding of the VLDL particle assembly process, which requires

apolipoprotein B. Unfortunately, there is a lack of direct structural information available

on apoB. Models based on the structure of a homolog (described below) have been helpful

but only solving the structure of apoB, or its domains, will reveal the structural features

that take part in this assembly process.

Apolipoprotein B

ApoB is one of the largest proteins in the body, composed of 4563 amino acids (513

kDa). There are two naturally occurring versions in humans: apoB48 and apoB100 (Figure

1.4). The numbers following the acronym refers back to the percentage of the molecule it

is composed of (i.e. apoB100 is 100% of apoB sequence and apoB48 is 48%). The region

of apoB that is homologous to lipovitellin is B 0% to 20%, and the secondary structure

and sequence of apoB20 are shown in Figure 1.4b and 1.4c. This same format will be used

to identify different constructs of apoB studied throughout the text. In humans, apoB100

is found only in lipoproteins secreted by the liver (VLDL and its metabolic byproducts

IDL and LDL) and apoB48 in chylomicrons that are secreted by the intestines and carry

9

fats from the diet to the peripheral tissues. The details of apoB assembly and its mecha-

nism of action are poorly understood, in large part because its tertiary structure remains

undetermined.

It is believed that apoB-containing lipoproteins start assembling co-translationally

[Hussain et al., 1998]. It has been shown that nascent apoB interacts with microso-

mal triglyceride transfer protein (MTP) and this interaction is crucial [Herscovitz et al.,

1991] [Hussain et al., 1998] to both lipid recruiting and proper folding. Our research group

developed the following model (Figure 1.5) to represent the assembly of apolipoprotein

B-containing lipoproteins. 1. First, a transient triglyceride lens (patch) is formed between

the two membrane leaflets by the action of diglycerol acyltransferase (DGAT) that pro-

duces TAG in the smooth endoplasmic reticulum. 2. The first 13% of apoB (apoB13)

exits the translocon. The β-barrel and α-helical domains bind to MTP (shown) and other

chaperones (not shown) [Zhang and Herscovitz, 2003] [Linnik and Herscovitz, 1998]. Part

of the α-helical domain (bound to MTP and chaperones) associates with the ER membrane

and the nascent protein gets anchored to it. 3. As the c-sheet emerges, its hydrophobic

side associates with lipids (lipid association shown in Herscovitz et. al., 1991). Note that

it is in this step that apoB9-17 (circled in red: dark blue region of the α-domain and the

red c-sheet domain) region is of particular relevance. It contains an MTP-binding region

in the α-helical domain and the lipid binding c-sheet domain. 4. B9-17 is secreted without

lipids, probably due to misfolding. 5. If the process is not terminated, other domains

(i.e. a-sheet and β1-domain) come in and create an envelope to finish building the nascent

VLDL particle. This nascent VLDL (also termed ”initiation particle”) is further lipidated

in an MTP-dependent fashion. As the model shows, the domains that are initially translo-

cated through the translocon are essential to recruit triglycerides into these lenses in the

ER membrane.

10

Figure 1.4: ApoB Structure and Sequence: ApoB is one of the largest proteins in the

body. With 4563 amino acids, it is more practical to refer to percentages of the protein

than specific amino acids. a. It is generally believed to have a pentapartite structure [Yang

et al., 1989] [Segrest et al., 1994b] with alternating alpha-helical and beta-sheet domains.

b. The major domains from the N-terminus of apoB (apoB20) are expanded, colored and

shown. This color scheme will be used throughout. ApoB9-17 contains residues 430 to 782

of human apo B. White box in c-sheet marks the region missing from the crystal structure

of lipovitellin (residues 675 to 738), believed to be an amphipathic alpha helix. c. ApoB20

sequence with signal sequence (first 27 residues, in gray - thus, residues 430 of 782 of the

mature protein correspond to residues 457 - 809 in this figure). ApoB9-17 is highlighted

using the same color scheme (cyan for alpha helix and red for beta sheet) to match the

predicted secondary structure.11

Figure 1.5: ApoB Secretion and Assembly Model: ApoB is an essential component

of the lipid metabolism pathway. Studies have shown that ApoB interacts with MTP to

achieve proper protein folding and to ultimately to recruit lipids. Further discussion of this

model is found in the text above.

The structure of LDL particles has been studied in several labs and models at more

than 20 A resolution have been proposed using cryo-electron microscopy (cryo-EM) and

crystallography [Orlova et al., 1999] [Spin and Atkinson, 1995] [Lunin et al., 2001] [Ren

et al., 2010] [Liu and Atkinson, 2011b] and [Liu and Atkinson, 2011a]. Based on these

low-resolution models, ApoB appears to form a belt that wraps around on the surface of

an LDL particle (Figure 1.6). Cryo-EM studies also indicate that the N-terminal region

of apoB protrudes away from the LDL particle, suggesting a globular fold (marked at top

right of Figure 1.6).

12

Figure 1.6: cryo-EM LDL Particle Reconstruction The larger volume in gray repre-

sents the overall shape of the LDL particle, which is overlaid with the high-density region

(orange) corresponding to the apoB portion. The overlaid structure was turned 90◦ in

each frame. The position of the N-terminal is indicated by triangles in the right and top

views [Liu and Atkinson, 2011b]

ApoB has been found to be homologous to a class of large lipoprotein transfer proteins

(LLTP) that include lipovitellin and MTP [Segrest et al., 1999]. The structure of lipovitellin

has been determined [Bradbury et al., 1999] [Thompson and Banaszak, 2002] [Hussain

et al., 2012] [Wu et al., 2013] and it is homologous to the N-terminal 20% of apoB (i.e.

apoB20) [Segrest et al., 1999] [Raag et al., 1988] [Anderson et al., 1998] [Mann et al.,

1999] [Thompson and Banaszak, 2002]. Several laboratories, including ours, have used this

structure to create models of the N-terminal region of apoB [Jiang et al., 2005] [Jiang et al.,

2007]. By fitting the first 20% amino acids in apoB into the crystal structure of lipovitellin,

we were able to obtain the model depicted in Figure 1.7. The model was tested by limited

proteolysis, chemical cross-linking and circular dichroism and domains matched the folding

proposed [Jiang et al., 2005]. These studies have validated the use of lipovitellin as a model

for the N-terminal of apoB. However these models lack sufficient detail for targeted drug

design.

13

Figure 1.7: Lipovitellin Homology Model: Lipovitellin homology model of apoB20

colored by domain (PDB 1LSH). It has been colored by domain: β-barrel in green (residues

1 - 292), α-helical domain in cyan (residues 293 - 593), c-sheet domain in red (residues 594 -

923) and the a-sheet region in dark blue (not relevant for this study). Silver beads (circled

in light yellow) represent possible lipid location.

Current methods to lower LDL include: cholesterol synthesis inhibitors, low-fat diets

and, more recently, antisense oligonucleotides, which inhibit apoB mRNA availability [Bell

et al., 2012] [Gebhard et al., 2013] [Phan and Toth, 2013]. Clinical targets for LDL levels

have recently become more aggressive and existing therapies are not sufficient to meet these

goals. Moreover, a number of patients have shown signs of intolerance to current therapies

[Golomb and Evans, 2008]. ApoB levels in plasma correlate directly with those of LDL,

and it has been identified as a promising therapeutic target [Lilly and Rader, 2007]. To

design selective drugs, the target has to be described in atomic detail. This is why solving

the structure of apoB is imperative to move forward the development of new therapies

to control LDL plasma levels. Additionally, it provides a base to understand diseases

caused by point mutations in apoB, like the one found in familial hypobetalipoproteinemia

(FHBL), R463W.

14

Most mutations resulting in FHBL are due to truncated forms of apoB that are cleared

more rapidly and thus, plasma levels of VLDL are abnormally low. FHBL is a rare condition

that impairs the body’s ability to absorb and transport fats as well as to absorb fat-soluble

vitamins. Individuals with this genetic defect are often unable to grow or gain weight at

the expected rate. More often than not, truncated forms of apoB are unable to form a

lipoprotein, causing accumulation of fat in the liver, which can lead to non-alcoholic fatty

liver disease [Schonfeld, 2003]. FHBL point mutation R463W is located on helix 10 in the

α-helical domain of apoB facing the c-sheet domain (Figure 1.8, yellow sphere). R463W is

one of only a few point mutations that result in FHBL [Burnett et al., 2003]. The mutation

to R463K restores near normal secretion of apoB from cell culture [Burnett et al., 2003].

This suggests that R463K forms a salt bridge to the helical domain, thereby orienting the

two domains to each other. This interaction between the helical domain and the c-sheet

domain and its dependence of R436 has been shown in vitro [Burnett et al., 2007].

Previous efforts to solve the structure have been unsuccessful, I hypothesize, because

they have focused on large constructs. In addition, apoB is known to be very hydrophobic

(especially the β2, α3, β4 and α5 regions). Lipovitellin models provide a foundation for

structural studies and have been helpful, but they do not provide the essential level of de-

tail. I am using a ‘divide and conquer‘ approach that aims to solve the structure of apoB

one piece at a time. This study presents apoB9-17 as a starting fragment to understand

and characterize key interactions of apoB, especially that with MTP.

15

Figure 1.8: ApoB20 Model (with apoB9-17 highlighted in darker colors): Same color

scheme as Figure 1.7. Darker colors highlight apoB9-17. In cyan, apoB residues 430 to

613 part of the alpha-helical domain. In red, apoB residues 614 to 782 part of the c-

sheet domain. Yellow sphere within the red region indicates the location of the mutation

(R463W) found in patient affected by Familial Hypobetalipoproteinemia (FHBL).

16

ApoB9-17 (Figures 1.9 and 1.4) contains helices 10-17 of the α-helical domain and first

6 β-strands of the c-sheet domain. Studies have shown that this region of apoB plays a

part in MTP association, necessary for VLDL particle assembly initiation process and also

is necessary for the lipid recruiting process [Herscovitz et al., 1991] [Hussain et al., 1998].

Limited proteolysis experiments done by our laboratory suggest that it will be a stable

construct, suitable for structural studies [Jiang et al., 2005].

This study aims to test the hypothesis that apoB9-17 is, in fact, suitable for structural

studies. If my hypothesis is correct, apoB9-17 will provide a model system for further

studies of apoB structure and function.

Figure 1.9: ApoB9-17 Predicted Structural Features and Functions: Three views

of the predicted structure of apoB9-17. Same color scheme as Figure 1.8 above. First

view looks at the interface between domains. Rotating 90◦ forward gives the second image.

Another rotation, this time of 180◦ into the page, result in opposite view and last image.

The predicted structure of apoB9-17 has a region of amphipathic α-helices (cyan) and a

region of amphipathic β-sheets, the c-sheet (red). Following this model, the α-helices face

the aqueous phase on the outer part of the lipoprotein. The β-sheets face the lipid core,

present a hydrophobic surface and are believed to be involved in the lipidation of VLDL.

17

Materials and Methods

2.1 Materials

2.1.1 Laboratory Equipment, Chemicals and Kits

CD Spectrometer Aviv Biomedical, Inc., AVIV 62 DS

Dialysis Membrane MW 10,000, Spectrum Laboratories 086806G

FPLC System GE Healthcare, AKTA

Gel Filtration Column Superdex 200 10/300 GL

Sonicator Branson SonicPower Co, W-350

UV-VIS Spectrophotometer PerkinElmer, Lambda 25

Protein Molecular Standards Novex LC5800, Sharp Pre-Stained Protein

STD

Purification Resin Thermo Scientific, His Pur Ni-NTA Resin

Chemiluminescence Kit Pierce, Super Signal West Femto substrate kit

2.1.2 Cells and Antibodies

Novagen BL21 E. coli bacterial expression cells

Primary Antibody (western blot) - PentaHis antibody, Biogen #34660

Secondary Antibody (western blot) - Flag monoclonal anti-Flag antibody, Sigma

2.1.3 Solutions and Buffers

Gel Filtration Buffer 20 mM phosphate, 150 mM NaCl, pH 7.0

Ponceau-s Staining Solution ST - 180, Boston BioProducts

Blocking buffer Superblock (PBS), Thermofisher

18

PBS Buffer 10X 0.03 M KCl, 1.4 M NaCl, 0.1 M

NaH2PO4·H20

Purification Buffers (The QIAexpressionist Kit Manual)

Lysis Buffer A 6 M Urea, 100 mM NaH2PO4, 10 M Tris·Cl,

20µM BME, pH 8

Washing Buffer B 8 M Urea, 100 mM NaH2PO4, 10 M Tris·Cl,

20µM BME, pH 8

Washing Buffer C 8 M Urea, 100 mM NaH2PO4, 10 M Tris·Cl,

20µM BME, pH 6.3

Elution Buffer D 8 M Urea, 100 mM NaH2PO4, 10 M Tris·Cl,

pH 5.9

Elution Buffer E 8 M Urea, 100 mM NaH2PO4, 10 M Tris·Cl,

pH 4.5

2.1.4 Software

BLAST Basic Local Alignment Search Tool, NCBI

K2D2 http://k2d2.ogic.ca

VMD Developed with NIH support by the Theoretical and

Computational Biophysics group at the Beckman Institute,

University of Illinois at Urbana-Champaign

19

2.2 Methods

2.2.1 Construct Expression

Transformation of BL21 Bacterial Cells

Transformation of bacterial cells was performed using standard procedures [Sambrook,

2001]. Briefly, calcium chloride competent BL21 (DE3) cells and B9-17 plasmid, pApoB9-

17, were thawed on ice for approximately 20-30 minutes. 1 µL of plasmid was added to 200

µL of thawed cells and mixed by flicking the bottom of the tube with a finger. The mixture

incubated on ice for ten minutes. After incubation, the mixture was heat shocked for 45

seconds at 42 ◦C. After heat shock, it was again incubated for five minutes on ice. 500 µL

of LB media at 37 ◦C were added to the cells and they were put into an incubator for one

hour at 37 ◦C to allow cells to express the kanamycin resistance gene on the pet24a-based

plasmid. After the incubation, 100 µL of the mixture were plated on four LB/Agar/Kan

(25 µg kan) plates and incubated overnight at 37 ◦C.

Induction of BL21 Bacterial Cells

Ten colonies were selected from each plate, to seed 10 mL of LB media containing 5µ/mL

[35µg/mL] kanamycin. After seeding, the mixture was incubated for 45 minutes at 37 ◦C.

While this was taking place, 4 X 2.8 L Fernbach flasks containing 1 L LB media with

kanamycin, each were placed to warm (37 ◦C) and stir in a floor shaker incubator. The

10 mL cultures were used to seed the 1 L flasks. The optical density at 600 nm (OD600)

was measured for four hours until a measurement of 0.4-1.0 was reached. 1 mL IPTG was

added to 1 mM final concentration to induce protein expression. After 2 hours of growth,

cultures were centrifuged at 10,000 x g for 20 minutes to harvest the cells. The pellet was

frozen at -20 ◦C until needed.

20

2.2.2 Construct Purification

Purification of ApoB9-17 via Ni-NTA Column

The frozen cell pellet was thawed and solubilized in 8 M urea, pH 8 with a ratio of 10 mL

per 1 gram of pellet. The mixture was sonicated in pulses for one minute, 5 times at low

power and rested on ice between pulses. The sample was incubated at room temperature

for 20 minutes to allow the urea to lyse cells and solubilize the apoB9-17. The sample

was then centrifuged for 20 minutes at 10,000 x g. The pellet fraction was washed with

2 mL lysis buffer A (pH 8) containing 20 µM β -mercaptoethanol (BME). The Ni-NTA

column was equilibrated with same buffer. The sample was then applied to the column

and incubated for 20 minutes to allow time for binding before setting the flow rate to 0.4

mL/min. The column was washed twice with 2 mL aliquots of buffer B (8 M Urea, 100

mM NaH2PO4, 10 M Tris·Cl, 20µM BME, pH 8) and C (8 M Urea, 100 mM NaH2PO4,

10 M Tris·Cl, 20µM BME, pH 6.3), then eluted with two aliquots of 2 mL of buffers D (8

M Urea, 100 mM NaH2PO4, 10 M Tris·Cl, pH 5.9) and E (8 M Urea, 100 mM NaH2PO4,

10 M Tris·Cl, pH 4.5), both without BME. All fractions were collected and frozen.

Dialysis

To remove the elution buffer’s high urea content from the purified fractions, they were

dialyzed. The purification fractions containing the apoB9-17 construct were thawed on ice

for one hour. The dialysis membrane (10,000 MW cutoff) was pre-washed with water. The

fractions were dialyzed against 1L 1X PBS buffer (diluted from 10X: 0.03 M KCl, 1.4 M

NaCl, 0.1 M NaH2PO4·H20) at 4 ◦C. The buffer was changed twice over the course of two

days. After dialysis, the sample was taken out of the membrane and centrifuged at 27,000

x g for 25 minutes to remove any particulates. Then the supernatant was divided threefold,

into aliquots of approximately 5 mL each. One part was lyophilized, another frozen at -20

◦C and another stored at 4 ◦C. 50 µL samples were taken for SDS page analysis (pellet

and solution) and circular dichroism (solution).

21

2.2.3 Construct Analysis

Western Blot

Proteins were separated by SDS-PAGE on a 4-20% gradient gel and transferred onto PVDF

(Immobilon-P) membrane at 80 V for 2 hrs. at room temperature in cold transfer buffer.

After rinsing with water, protein bands were detected with a Ponceau-S stain. After wash-

ing, the membrane was incubated with gentle agitation throughout all subsequent steps.

Membrane was blocked in Superblock (PBS) blocking buffer (ThermoFisher) for 1 hr at

room temperature to reduce non-specific binding. Membrane was then incubated with Pen-

taHis antibody (Biogen) (diluted 1:1000 in Superblock) overnight at 4 ◦C. The membrane

was washed for 10 min. 3X with washing buffer (TBS / 0.1% Tween-20) at room temper-

ature. The membrane was incubated with horseradish peroxidase-conjugated secondary

antibody (diluted 1:5000 in Superblock) for 1 hr. at room temperature. The membrane

was extensively washed 3-4 times for 10 min. each at room temperature with washing

buffer (TBS / 0.1% Tween-20). The protein bands were detected by chemiluminescence

using the Super Signal West Fempto substrate kit (Pierce).

Circular Dichroism Spectra

Circular dichroism measurements were performed on an AVIV 62DS spectrometer using

quartz cuvettes of 1 mm path length. Scans between 185 and 250 nm were performed at

a scan speed of 12 nm/min, bandwidth of 1.0 nm, a 5 s average time, and reported as

an average of three scans. The quartz cuvette temperature was controlled (25 ◦C) with a

Peltier temperature controller. CD spectra of apoB9-17 were measured in 1X PBS and the

baseline corrected against buffer alone. Construct concentration was approximately 9.21

µM (0.456 mg/mL).

22

Calculations for Concentration Determination

The sample used in CD experiments was analyzed by UV-Vis spectroscopy to determine

the concentration of the protein in solution. Approximately 1 mg of lyophilized protein

sample was solubilized in 1 mL 1X PBS buffer. The absorbance at 280 nm was measured

with a Perkin-Elmer, Lambda 25 UV-Vis Spectrophotometer.

The concentration was calculated using Beer-Lambert Law:

A = εlc (2.1)

where,

A = absorbance at 280,

ε = extinction coefficient = 24,300,

l = path length = 1 cm,

c = unknown concentration,

2.2.4 Size Exclusion Chromatograpy

Purified apoB9-17 was analyzed by size-exclusion to determine its effective molecular weight

in purified solution. Samples were loaded on to the column which was equilibrated with

PBS and eluted at a flow rate of 0.5 mL/ min. The absorbance was monitored at 210 and

280 nm. The fractions corresponding to the apoB9-17 peak were collected and pooled. The

molecular mass of the complex was calculated based on the elution of reference proteins

thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 k)Da, lactate dehydrogenase (140

kDa) and bovine serum albumin (66 kDa) (GE healthcare, USA). The ratio of the retention

volume of the calibration proteins and the void volume were plotted versus the logarithm

of the molecular weight of the proteins, and linear regression was used to calculate the

molecular weight of the complexes (not shown).

23

Results

3.1 Expression and Purification of ApoB9-17

To produce the apob9-17 construct, bacterial cells were transformed with a constructed

plasmid. The plasmid previously constructed by Andrew Bogorad and Jacob Turnbull

confers kanamycin resistance. The apoB9-17, residues 430 to 782 of human apoB (shown

in Figure 1.4) is followed by 6X-His tag. Novagen BL21 cells expressed large amounts of

protein, making them a good choice for an expression system for the study. Typical pro-

tein expression levels can be seen in Figure 3.1 (lane 3), measured 2 hours after cells were

induced with IPTG. Cells were harvested by centrifuging the induced samples at 1580 x

g for 20 minutes. The pellet and the supernatant were separated and both samples were

frozen at -20 ◦C.

To determine if the protein was being produced in soluble or insoluble (inclusion bodies)

form, the pellet was processed further. The pellet was resuspended in 30 mL of buffer B (8

M Urea, 100 mM NaH2PO4, 10 M Tris·Cl, 20µM BME, pH 8 (HCl)). It was then sonicated

by intervals at 3X power, 5 times, resting on ice in between. The solution was allowed to

sit for 20 minutes and then the sample was centrifuged at 10,000 x g for 20 minutes. The

supernatant was separated from the pellet. The pellet fraction was re-suspended in the

original volume (30 mL) of buffer B. Samples were taken from both fractions and analyzed

by SDS-page. Figure 3.2 (arrow) shows that the construct prefers the pellet fraction, re-

siding in inclusion bodies (insoluble fraction). Fractions then were frozen at -20 ◦C until

purification.

24

Figure 3.1: ApoB9 - 17 construct is expressed well in BL21 cells. Lanes contain

(from left to right): 1) the molecular weight marker, 2) sample taken just previous to protein

induction with 1 mL (1µL/M) IPTG and 3) sample taken 2 hours after induction. The

SDS-PAGE gel was stained with coomassie blue. The arrow indicates apoB9-17 construct.

25

Figure 3.2: ApoB9-17 construct is found in the insoluble fraction after expres-

sion. Lanes contain (from left to right): 1) Molecular weight standards, 2) sample of

solution after centrifugation and, 3) sample of pellet after centrifugation. The SDS-PAGE

gel was stained with Coomassie blue. ApoB9-17 is in the pellet fraction as indicated by

the arrow.

26

To ensure the major band visible by Coomassie staining was in fact apoB9-17, a west-

ern blot assay using anti 6-His antibody was performed. The western blot (Figure 3.3,

arrow) confirms successful expression of the full protein. The protein band falls within the

expected molecular weight value of 39 kDa. Additionally, recognition of the c-terminal by

a single band in the western blot confirms the construct is resistant to proteolysis.

Figure 3.3: Western Blot confirms the identity of ApoB9-17 in the pellet fraction.

Lanes contain (from left to right): the molecular weight marker, sample taken just previous

to protein induction with 1 mL (1µL/M) IPTG, sample taken 2 hours after induction and

resulting fractions of a centrifuged induced sample (pellet and solution fractions are shown).

ApoB9-17 is mainly in the pellet fraction as indicated by the arrow.

27

After solubilizing the pellet with 8 M urea and re-centrifugation, the lysate was purified

by means of a Ni-NTA affinity column. Purification is shown in Figure 3.4. Lane 1 shows

the sample prior to loading on the Ni-NTA beads. The sample was then loaded into the

column and after mixing for 30 minutes, allowed to flow slowly out of the Ni-NTA column.

The flow through was re-applied to the column and allowed to flow through without any

wait or further mixing. Then the column was washed twice with buffer B (8 M Urea, 100

mM NaH2PO4, 10 M Tris·Cl2O, µM BME, pH 8) and twice with buffer C (8 M Urea, 100

mM NaH2PO4, 10 M Tris·Cl, 20µM BME, pH 6.3) after that (Lanes 4 - 7). Both washes

contained BME, a reducing agent, to prevent disulfide bonding during purification and the

formation of multimeric chains. The second wash, buffer C, had a pH of 6.3. which is lower

than the binding buffer (B, pH 8), this allowed to remove non-specifically bound proteins.

Then, the protein was then eluted 3 times with buffer D (8 M Urea, 100 mM NaH2PO4,

10 M Tris·Cl, pH 5.9) and 3 times with buffer E (8 M Urea, 100 mM NaH2PO4, 10 M

Tris·Cl, pH 4.5) (Figure 3.4 lanes 8 - 11). The pH of these buffers gradually lowered disrupt

the coordination bond between the histidine and the nickel by protonation of the imidazole

nitrogens. ApoB9-17 is visible in lanes 10 and 11 (indicated by the arrow) as well as lane

2. A single band, of approximately 39 KDa, can be observed on lanes 10 and 11, indicating

successful purification. Fractions 10 - 11 were pooled and then dialyzed into PBS to remove

urea.

28

Figure 3.4: Purification of ApoB9-17: Tricine gel, stained with Coomassie blue, showing

purification of the apoB9-17 construct by means of a Ni-NTA column. Lane 2 shows the

sample prior contact with the Ni-NTA beads. Lanes 4 - 7 are washes and lanes 8 - 11 are

elutions. ApoB9-17 is visible in lanes 10 and 11 (indicated by the arrow).

29

3.2 Spectral Properties of ApoB9 - 17

Analysis of spectral properties can provide more detail of the structure at hand. Circular

dichroism (CD) can be used to determine the secondary structure of proteins. Far-UV

CD spectra of an undiluted solution of purified apoB9-17, dialyzed into PBS, shows a

mostly alpha helical conformation (Figure 3.5). Further analysis by K2D2 structure pre-

diction tool shows the structural composition to be roughly 76% α-helical and 2% β-strand.

To test if apoB9-17 is folded cooperatively as expected, the signal at 222 nm that indi-

cates helical structure was monitored as a function of temperature. Figure 3.6 shows that

between 50 ◦C and 70 ◦C the protein largely unfolds. The solution after thermal unfold-

ing contained visible precipitate (not shown). After removing the precipitated protein by

centrifugation, the CD scan before thermal denaturation (Figure 3.5) and CD scan after

thermal denaturation (not shown) exhibit the same shape and thus, secondary structure.

From the data obtained, it was determined that apoB9-17 is stable at room and physio-

logical temperatures, and folding is reversible, up to the temperature where it aggregates.

30

Figure 3.5: ApoB9-17 Secondary Structure Determination by CD Spectra. CD

Spectra obtained of ApoB9-17 at 11µM protein in 1 mm path length cuvette, pH 7, 25 ◦C.

Spectrum is average of 3 scans.

31

Figure 3.6: Thermal denaturation monitored by Circular Dichroism. 11 µM B9-17,

pH 7, 1 mm path length, heated in 2 deg steps with a 1 min equilibration time, monitored

by the CD signal at 222 nm.

32

3.3 Aggregation State of ApoB9-17

The effective molecular weight of apoB9-17 was analyzed by size exclusion chromatography.

As shown is Figure 3.7, a single peak can be observed at 14.12 mL. After analysis of the

standards, we obtain an estimated molecular weight of 89 kDa. This is roughly double the

expected molecular weight for a single apoB9-17 molecule (39 kDa). This indicates that

apoB9-17 exists as a single dimeric species in solution.

A possible explanation for this lies in the projected structure, Figure 1.8. The portion

of the c-sheet, proposed to be in close proximity to lipids and be part of the lipid recruiting

process (Figure 1.5), can potentially be associating with a c-sheet from another monomer

of apoB9-17. Potentially, disruption of this association could be achieved by addition of

mutations to the β-strands on the c-sheet. For example, the mutation of hydrophobic

residues into charged residues in the c-sheet.

33

Figure 3.7: ApoB9-17 forms a single, dimeric species. ApoB9-17 is a single species

by size exclusion chromatography. The column was a superdex 200 column run at 0.5 ml/

min in 20 mM phosphate, 150 mM NaCl, pH 7.0, 25 ◦C. Molecular weight standards are

shown in grey (k = kDa), along with their respective molecular weights.

34

Conclusions

Atherosclerotic plaques are the underlying cause of death of more than 600,000 Americans

each year. High levels of low-density lipoprotein (LDL) in the bloodstream have been

identified as the main culprit for the development of these plaques. There are a few

therapies available that control LDL levels to some extent. Nevertheless, patients in need

of aggressively low LDL levels cannot be treated effectively with these therapies. In order

to advance the medical field, new therapeutical targets need to be identified.

One promising target is to regulate the secretion of apolipoprotein B (apoB) and lipid-

recruiting process. The experiments described in this thesis aim to help advance structural

determination of apolipoprotein B. Through this research, we now have a fragment of apoB

(apo9-17) that has potential to be studied further.

The findings from this thesis show that apoB9-17:

• Can be successfully produced in large (milligrams) quantities via BL21 expression.

• Ni-NTA column purification successfully separates the target construct.

• Gel filtration results show that apoB9-17 exists as a single dimeric species in solution.

The dimer may formed by hydrophobic interactions between the c-sheet from one

apoB monomer and the c-sheet of another apoB monomer.

• Far-UV CD spectra show a secondary structure that agrees with the homology mod-

els of apoB and the region 9-17. Using CD data, software prediction of secondary

structure estimates the structure is about 76% α-helical and 2% β-sheet.

• CD scans after thermal denaturation experiments show that the protein construct

still in solution retains its secondary structure. Consequently, the protein is stable

35

at room and physiological temperatures. Precipitation during thermal denaturation

maybe due to high protein concentrations.

Previous approaches to elucidate apoB structure have not been successful, mainly due

to the protein’s large size and the analysis approach. By targeting the protein one section

at a time, we will get answers in a staggered way, that is likely to be successful and faster.

The findings of this study are a key step in this ”divide and conquer” approach.

The region of apoB9-17 is thought to be closely related to the formation of the LDL

particle. As seen in our model, Figure 1.5, apoB associates with MTP and recruits lipids

to form the LDL particle. Moreover, the model also shows that the section discussed in

this study, 9-17, includes the portion of apoB that initially associates with MTP and lipids.

Thus, determination of apoB9-17 structure can help pin point that region of MTP and lipid

affinity which consequently can help find a way to control apoB assembly and ultimately

manipulate LDL particle formation.

Furthermore, apoB 9-17 is the setting for the naturally-occurring point mutation (R463W)

that is present in familial hypobetalipoproteinemia (FHBL). FHBL is a genetic disease

that reduces apoB secretion. Misfolded apoB is cleared in the endoplasmic reticulum by

ubiquitination and the proteasome, or via autophagy. Thus, plasma levels of VLDL are

abnormally low. Unfortunately, the inability to secrete apoB can cause accumulation of

fat in the liver, which can lead to non-alcoholic fatty liver disease [Schonfeld, 2003]. FHBL

point mutation R463W is located on helix 10 in the α-helical domain of apoB facing the

c-sheet domain. This further justifies the selection of this region of apoB as a starting

point, in order to determine the effect of this mutation, which is expected to be buried

between the helical domain and the c-sheet, in the apoB9-17 structure.

The evidence presented here, indicates that apoB9-17 is a suitable construct for struc-

36

tural and functional analysis. In the future, the apoB9-17 structure can be studied by

x-ray crystallography. Concurrent with x-ray diffraction experiments, 15N and 13C het-

eronuclear single quantum coherence (HSQC) spectroscopy experiments could be useful to

screen for unfolded regions. Other NMR experiments can aid to elucidate the structure by

providing restrictions from which are analyzed with CNS to create electron density maps.

Also NMR experiments can provide sequential connectivity between residues, which allows

us to link the residues previously assigned.

The construct, which the experiments here outlined, show that it is purified as a dimer.

A potential further direction would be to design a monomeric form of apoB9-17 by chang-

ing hydrophobic residues to polar residues on the c-sheet. Once the structure has been

determined, the spectra can be compared to that of constructs containing mutations such

as R463W and of the protein interacting with lipids. This can help further the model

developed by the laboratory.

Additional biophysical properties can be studied using drop tensiometry. Drop ten-

siometry can be used to study the construct’s interfacial behavior of apoB9-17 at the

lipid-interacting interface that mimics the luminal leaflet of the ER membrane. It can

measure surface pressure at the interface under the presence of various modifiers like: fatty

acids and cholesterol. Coupled with binding assays, we can learn more about the con-

struct’s lipid affinity.

NMR spectra of a triple-labeled apoB9-17 in the presence of MTP can test protein-

protein interface. The data from these experiments can be used to understand that interface

from a structural perspective.

Thus, this information opens up the door to further the knowledge on apolipoprotein

B and in turn, give way to new therapies for various cardiovascular diseases.

37

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Curriculum Vitae

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