Structural folding dynamics of an archetypal conformational disease using Nuclear Magne

214 Pages·2014·21.65 MB·English

Structural
 folding
 dynamics
 of
 an
 archetypal
 conformational

disease
 using
 Nuclear
 Magnetic
 Resonance
 Spectroscopy

Ph.D.
 Thesis

Géraldine
 Rosalie
 Levy

Institute
 of
 Structural
 and
 Molecular
 Biology
 (ISMB)

University
 College
 London

Submitted
 September
 2013
Declaration

I,
 Géraldine
 Rosalie
 Levy,
 declare
 that
 all
 the
 work
 presented
 in
 this
 thesis
 is

the
 result
 of
 my
 work
 only.
 Where
 information
 has
 been
 derived
 from
 other

sources,
 I
 confirm
 that
 this
 has
 been
 indicated
 in
 the
 thesis.
 The
 work
 herein
 was

carried
 out
 while
 I
 was
 a
 post-­‐graduate
 student
 at
 University
 College
 London,

Institute
 of
 Structural
 and
 Molecular
 biology,
 under
 the
 supervision
 of
 Dr
 John

Christodoulou
 and
 Dr
 Bibekbrata
 Gooptu.

  1
Acknowledgements

  2
Abstract

Members of the serpin (serine protease inhibitor) superfamily of proteins regulate key
physiological processes through their ability to undergo major conformational
transitions. In conformational diseases, native protein conformers convert to
pathological species that polymerise. Structural characterization of these key
transitions is challenging. Mechanistic intermediates are unstable and minimally
populated in dynamic equilibria that may be perturbed by many analytical techniques.
I use Nuclear Magnetic Resonance (NMR), and Circular Dichroism (CD)
spectroscopy, to investigate the interrelated processes of serpin folding, misfolding
and polymerisation in solution using the 45kDa prototypic serpin α -antitrypsin, the
1
recent assignment of the backbone resonances of α -antitrypsin by our group, allows
1
us to ask more sophisticated questions by a range of NMR techniques to study its
structure and dynamics. In this study, I analysed early unfolding behaviour of α -
1
antitrypsin across a urea titration within what is apparently the largest two-states
system yet characterised. In order to assess the dynamics of the native state, I have
used hydrogen/deuterium exchange nuclear magnetic resonance spectroscopy
(HDXNMR) to characterise motions on the slow (ms) timescale. I have conducted a
detailed analysis of residue-specific changes in protection from exchange across a pH
titration using SOFAST-HMQC. This is complemented by a detailed a preliminary
analysis of fast motions (ps-ns) using NMR relaxation experiments. Moreover, a
forme fruste deficiency variant of α -antitrypsin (Lys154Asn) that forms polymers
1
recapitulating the conformer-specific neo-epitope observed in polymers that form in
vivo was characterized in this study. Lys154Asn α -antitrypsin populates an
1
intermediate ensemble along the polymerisation pathway at physiological
temperatures. Together, this study shows how the use of powerful but minimally
perturbing techniques, mild disease mutants, and physiological conditions, provides
novel insights into pathological conformational behaviour.

  3
List
 of
 abbreviations

Serpin:

  Serine
 Protease
 Inhibitor

FENIB:

  Familial
 Encephalopathy
 with
 Neuronal
 Inclusion
 Body

NMR:

  Nuclear
 Magnetic
 Resonance
 spectroscopy

SDS:

  Sodium
 Dodecyl
 Sulfate

PAGE:

  Polyacrylamide
 Gel
 Electrophoresis

LB:

  Luria
 Broth

E.
 coli:

  Escherichia
 coli

Cryo-­‐EM:

  Cryo
 electron-­‐microscopy

CD:

  Circular
 Dichroism

RPM:

  Revolutions
 Per
 Minute

OD:

  Optical
 Density

PEG:

  Poly
 Ethylene
 Glycol

TUG:

  Transverse
 Urea
 Gel

βME:

 β
 Mercapto-­‐Ethanol

EDTA:

  EthyleneDiamineTetracetic
 Acid

IPTG:

  Isopropyl
 β-­‐D-­‐1-­‐thiogalactopyranoside

ER:

  Endoplasmic
 Reticulum

HSQC:

  Heteronuclear
 Single
 Quantum
 Coherence

TROSY:

  Transverse
 Relaxation-­‐Optimized
 Spectroscopy

RDC:

  Residual
 Dipolar
 Coupling

HMQC:

  Heteronclear
 Multiple
 quantum
 coherence

SOFAST:
  Selective
 Optimized
 Flip
 Angle
 Short
 Transient

DSS:

  4,4-­‐dimethyl-­‐4-­‐silapentane-­‐1-­‐sulfonic
 acid

MW:

  Molecular
 Weight

RNA:

  Ribonucleic
 Acid

  4
mRNA:

  Messenger
 Ribonucleir
 Acid

tRNA:

  Transfer
 Ribonucleir
 Acid

DNA:

  Deoxyribonucleic
 acid

cDNA:

  Complementary
 DNA

v/v:
 volume:

 volume
 ratio

w/v:
 weight:

 volume
 ratio

kDa:

  kilo
 Daltons

Δν :

  Linewidth

1H
τ :

  Rotational
 correlation
 time

C
RCL:

  Reactive
 Centre
 Loop

MS:

  Mass
 Spectrometry

IM/MS:

  Ion
 Mobility
 Spectrometry
 -­‐
 Mass
 Spectrometry
 experiments

ToF:

  Time
 of
 Flight

m/z:

  mass:charge
 ratio

CID:

  Collision
 Induced
 Dissociation

HDXNMR:

  Hydrogen
 Deuterium
 Exchange
 Nuclear
 Magnetic
 Resonance

Spectroscopy

HDXMS:

  Hydrogen
 Deuterium
 Exhange
 Mass
 Spectrometry

FID:

  Free
 Induction
 Decay

NOE:

  Nuclear
 Overhauser
 Effect

FT:

  Fourier
 Transformation

PDB:

  Protein
 Data
 Base

ω :

  Precession
 rate

0
γ:

  Gyromagnetic
 ratio

F Apparent
 fraction
 unfolded

app:

WT:

  Wild
 Type

∆G:

  Gibbs
 Free
 energy

∆H:

  Difference
 in
 enthalpy

T:

  temperature

  5
∆S:

  Difference
 in
 entropy

k :

  Rate
 of
 exchange

ex
k :

  Intrinsic
 rate

int
k :

  Rate
 of
 opening

op
k :

  Rate
 of
 closing

cl
∆G :

  Free
 energy
 between
 the
 close

app
GdmCl:

  Guanidine
 Hydrochloride

K:

  Equilibrium
 constant

F:

  Folded

U:

  Unfolded

I:

  Intermediate

M*:

  Molten
 Globule-­‐like

CFTR:

  Cystic
 Fibrosis
 Transmembrane
 Conductance
 Regulator

ERAD:

  ER-­‐associated
 degradation

EOR:

  ER
 Overload
 Response

S:

  Stressed

R:

  Relaxed

B Externally
 applied
 magnetic
 field

0:

R Transverse
 relaxation
 rate

2:

τ Rotational
 correlation
 time

c:

pET:

  pTermat
 Expression
 vector
 system

PCR:

  Polymerase
 chain
 reaction

AmPS:

  Ammonium
 persulphate

IM-­‐MS:
  Ion
 Mobility
 -­‐
 Mass
 Spectrometry

DTT:

  Dithiothreitol

U.V.:

  Ultra
 Violet

DSC:

  Differential
 Scanning
 Calorimetry

mAb:

  Monoclonal
 antibody

P:

  Protection
 factor

  6
IEF:

  Iso-­‐Electric
 Focusing

SI:

  Stoichiometry
 of
 Inhibition

K :

  Association
 rate
 constant

app
PAI-­‐1:

  Plasminogen
 Activator
 Inhibitor-­‐1

CCS:

  Collision
 Cross
 Section

INEPT:

  Insensitive
 Nuclei
 Enhanced
 by
 Polarization
 Transfer

  7
Table
 of
 Contents

Declaration
  1

Acknowledgements
  2

Abstract
  3

List
 of
 abbreviations
  4

Chapter
 1
 –
 INTRODUCTION
  12

Protein
 folding
  13

Physical
 forces
 and
 principle
 underlying
 protein
 folding
  13

1.1.1.1.
 Thermodynamics
  14

1.1.1.2.
 Kinetics
  16

1.1.1.3.
 Models
 of
 protein
 folding
  16

1.1.1.4.
 Polypeptide
 chain
 behaviour
 and
 energy
 landscapes
  18

1.1.1.5.
 Tools
 for
 the
 study
 of
 protein
 folding.
  20

1.1.1.6.
 Relationship
 of
 polypeptide
 folding
 within
 cells
 and
 in
 isolation
  20

1.1.1.7.
 Protein
 misfolding
 and
 conformational
 disease
  21

1.2.
 Serpins
 and
 serpinopathies
  23

1.2.1.
 α -­‐antitrypsin
 deficiency
  24

 1
1.2.2.
 Metastability
 and
 inhibitory
 mechanism
  25

1.2.3
 Alternative
 serpin
 conformations
  27

1.2.4.
 Studies
 of
 serpin
 folding
  29

1.2.5.
 Serpin
 polymerisation
  31

1.2.5.1.
 Polymerisation
 induced
 by
 loop
 cleavage
 or
 peptide
 insertion
  32

1.2.5.2.
 Three
 models
 of
 polymerisation
 proposed
 for
 disease
 mechanism
 and

intermediate
 formation
  34

1.3.
 NMR
 spectroscopy
 in
 studying
 protein
 folding
  38

1.3.1.
 General
 principles
 of
 NMR
  38

1.3.2.
 Study
 of
 protein
 structure
 and
 dynamics
 using
 NMR
 spectroscopy
  41

1.3.3.
 NMR
 studies
 of
 high
 molecular
 weight
 proteins
  42

1.3.4.
 Introduction
 to
 NMR
 of
 α-­‐antitrypsin
  46

1
1.3.5.
 Multiple
 timescales
 allow
 structural
 and
 dynamical
 behaviour
 to
 be
 characterized
 48

Chapter
 2
 –
 MATERIALS
 AND
 METHODS
  54

2.1
 Materials
  55

2.1.1
 Regents
  55

2.1.2
 Instruments
  55

2.1.2.1.
 Cell
 purification
 and
 characterization
  55

2.1.2.2.
 Circular
 Dichroism
  56

2.1.2.3.
 Nuclear
 Magnetic
 Resonance
 (NMR)
 Spectroscopy
  56

2.1.2.4.
 Mass
 Spectrometry
 (MS)
  56

2.2
 Methods
  57

2.2.1
 Protein
 recombinant
 expression
 in
 Escherichia
 coli
 (E.
 coli)
 and
 purification
  57

  8
2.2.1.1.
 Plasmid
 constructs
  57

2.2.1.2
 Transformation
 of
 BL-­‐21-­‐Gold
 E.
 coli
 with
 plasmid
 pQE-­‐31
 containing
 α-­‐antitrypsin

1
cDNA
  59

2.2.1.3
 Unlabelled
 recombinant
 α-­‐antitrypsin
 protein
 production
  59

1
2.2.1.4
 Uniform
 incorporation
 of
 15N-­‐
 and
 13C-­‐15N-­‐2H-­‐
 isotopic
 labeling
 for
 NMR
  60

2.2.2
 Purification
 of
 α-­‐antitrypsin
  60

1
2.2.3
 Biophysical
 and
 biochemical
 characterization
  62

2.2.3.1
 Activity
 Assay
  62

2.2.3.2
 Far
 U.V.
 Circular
 Dichroism
 spectroscopy
  63

2.2.3.3
 Native-­‐PAGE
  63

2.2.3.4
 Transverse
 Urea
 Gradient-­‐PAGE
  64

2.2.3.5
 1H
 NMR
  65

2.2.3.6
 Native
 Mass
 Spectrometry
 (MS)
  65

2.2.4
 Hydrogen
 Deuterium
 exchange
 (HDXNMR)
  66

2.2.4.1
 Sample
 preparation
  66

2.2.4.2
 1H-­‐15N
 SOFAST
 HMQC
 NMR
 for
 HDXNMR
 experiments
  66

2.2.4.3.
 Spectral
 processing
 and
 NMR
 frequency
 referencing
  67

2.2.4.4.
 Data
 processing
 and
 analysis
  68

2.2.5.
 NMR
 studies
 of
 α-­‐antitrypsin
 in
 urea
  70

1
2.2.5.1.
 Sample
 preparation
  70

2.2.5.2
 1H-­‐15N
 -­‐TROSY
 HSQC
  71

2.2.6.
 NMR
 relaxation
 of
 α-­‐antitrypsin
  71

1
2.2.6.1
 Sample
 preparation
  71

Chapter
 3
 –
 NMR
 STUDIES
 OF
 α-­‐ANTITRYPSIN
 SOLUTION
 BEHAVIOUR
 IN
 UREA
  74

1
3.1.
 Introduction
  75

3.1.1.
 Metastability/Z-­‐mutant
 and
 formation
 of
 intermediate
 ensemble.
  75

3.1.2.
 Equilibrium
 folding
 intermediate
  76

3.1.3.
 Probing
 the
 folding/unfolding
 pathway
 of
 α-­‐antitrypsin
 in
 guanidine
  78

1
3.1.4.
 Probing
 the
 folding/unfolding
 pathway
 of
 α-­‐antitrypsin
 in
 urea
  80

1
3.1.5.
 Effect
 of
 stabilizing
 ‘cavity-­‐filling’
 mutations
 on
 the
 native
 state
 α-­‐antitrypsin
  81

1
3.2
 Results
  82

3.2.1.
 NMR
 studies
 of
 α-­‐antitrypsin
 unfolding
 structural
 behaviour
  82

1
3.2.2.
 Circular
 Dichroism
 studies
 of
 the
 plasma
 derived
 and
 recombinant
 α-­‐antitrypsin
  92

1
3.2.3.
 Transiently
 populated
 α-­‐antitrypsin
 intermediates
 form
 off-­‐pathway
 polymers
 with

1
defined
 secondary
 structure
 in
 urea
  95

3.3.
 Discussion
  97

Chapter
 4
 –
 STRUCTURAL
 DYNAMICS
 OF
 THE
 NATIVE
 α-­‐ANTITRYPSIN
 STUDIED
 BY

1
HYDROGEN
 DEUTERIUM
 EXCHANGE
 NUCLEAR
 MAGNETIC
 RESONANCE
 SPECTROSCOPY

  101

4.1.
 Introduction
  102

4.1.1.
 General
 principle
 of
 HDXNMR
  102

4.1.2.
 HDXMS
 studies
 of
 α-­‐antitrypsin
  105

1

  9