Exploring Pepsin's Alkaline instability via Bioinformatic Analysis and a Rationale Protein Design

179 Pages·2017·16.23 MB·English

Exploring Pepsin’s Alkaline instability via Bioinformatic Analysis
and a Rationale Protein Design approach

by

Douglas Stuart Alexander Grahame

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Food Science

Guelph, Ontario, Canada

© Douglas Stuart Alexander Grahame, March, 2017
ABSTRACT

EXPLORING PEPSIN’S ALKALINE INSTABILITY VIA BIOINFORMATIC
ANALYSIS AND A RATIONALE PROTEIN DESIGN APPROACH

Douglas Stuart Alexander Grahame Advisor:
University of Guelph, 2016 Professor Rickey Y. Yada

Residues and motifs defining to the alkaline instability of pepsin and alkaline stability of renin have, as
of yet not been identified. To accomplish said task the present study utilized literature and a comparative
bioinformatic analysis to generate structural and functional information regarding protein stability, protein
folding, and mutational stability related to alkaline stability.
Site-directed mutagenesis was employed to generate 15 mutants in the N-terminal domain expected to
increase the alkaline stability of pepsin. Mutants D159L and D60A increased alkaline activity (P≤0.05)
whereas E4V and H53F were found to retain native structure at elevated pH levels (P≤0.05). Alleviating
charge – charge repulsion of carboxyl groups of interest was insufficient to increase the alkaline stability
of pepsin. The importance of the β-barrel was highlighted as 92% of the stabilizing residues identified by
SRide in the N-terminal lobe were located in β-structure of pepsin and renin. The comparative
bioinformatic analysis identified structure and sequence differences between pepsin and renin in β-strands
and turn/loop regions of the N-terminal lobe known to play a role in the alkaline denaturation of pepsin.
Differences in residues promoting acidic or alkaline stability were identified in pepsin and renin.
Flexibility and rigidity differences were identified where renin was found to be a more rigid protein with
specific areas of flexibility whereas pepsin has a higher level of general flexibility.
In summary, alkaline activity and stability was improved by reducing electrostatic repulsion of Asp159.
The ability of Asp159 to improve activity (P≤0.05) denotes electrostatics do play some role in alkaline
denaturation. However, overall alkaline activity and structural stability was not dramatically improved for
any of the mutants generated. Thus, alkaline stabilization of pepsin will require more than the point-
mutation reduction of electrostatic repulsions. The presence of stabilizing residues within β-barrel
structure implicates the hydrogen-bonding network of the β-barrel as important in defining stability.
Finally, flexibility differences and amino acid composition indicate that the increased rigidity of β-sheets,
the ψ-loop, and turn regions likely play a defining role in the alkaline stability difference between renin
and pepsin, and that stabilization occurs at a motif level rather than at a residue level.
ACKNOWLEDGEMENTS
After spending a decade in post-secondary institutions one would think that writing a single page
thanking all my friends and family would be an easy task. The truth of it is that this is the last page I
wrote for my thesis precisely because, for me, it was the hardest. I’ve read hundreds of different quotes
but none of them seemed to resonate with how thankful I am for all of the help I’ve received. What does
seem to resonate is that I didn’t say thank you enough during the process. So to try and make up for lost
time with the expectation that few to none of the people I need to and want to thank will ever actually see
this I’ll put it down on paper in some vain attempt to make up for lost time.
To all my friends back home, you always made it feel as if time and distance hadn’t changed our
friendship and your support and encouragement was not only important to help me along the way but
something I’ve treasured intensely. To all of the Windsor & Kinston crew, I know we don’t get a chance
to talk often but I can’t tell you how much I value every one of the friendships I was able to make during
these years. I don’t think I would have ever thought about to grad school if it wasn’t for things like grilled
cheese in Dillon Hall, Italian dinner time, the Campus Rec crew, and of course the power of the DNA
study group! For the OT crew thank you for taking me in as one of your own and if I may borrow your
tag line, it’s a celebration……As for Saskatoon, it was a dark, cold and an unforgiving place when we
first moved there that literally brought my wife to tears. We could not have been more wrong about a
place. To my fellow grad students, the ball hockey guys, CBI crew, and everyone else you made it hard to
leave and we miss you all dearly! Finally Guelph, just when I thought life couldn’t be filled with any
more amazing people we had the good fortune of meeting so many more of you in Guelph. After getting
married, having our first child, and of course finishing the PhD I doubt very much this place and all of
you who made it so special will ever be very far from my heart.
To all my lab mates over the years, I did it, I made it out, and it is possible! Jack, Hayley, Tae Sun,
Reena, Huogen, Jenny, John and of course the brain Brian himself thank you for all your help along the
way. Through beers, tears, thoughts of quitting, science rants, and late night talks I’ve had some of my
best memories with you all and I hope to hear from you all and all about your success for years to come!
iv
To all my teachers along the way and to my supervisors Dr. Warner, Dr. Tanaka, and Dr. Yada
specifically, I owe you a great debt for all the energy and countless hours you have spent on me. To Dr.
Warner, you took me in and gave me my first chance and I will always be thankful for giving me my first
shot and helping me find the path I wanted to follow. To Dr. Tanaka, your patience, kindness, support,
and friendship allowed me to explore, fail, learn, and succeed in ways I never thought possible. You gave
me the opportunity to build the foundation of my knowledge that my success sits on today. Thank you for
everything Dr. Tanaka! To Dr. Yada, you gave me an opportunity to achieve a goal that I have had for my
entire life and for that I will never be able to thank you enough. Your boundless support and inspiration
have meant the world to me let alone what you have taught me regarding science, career, and life along
the way. I am honoured to be a part of the Empire were I saw amazing things accomplished due to your
support and faith in an amazing, intelligent, and talented team. Thank you Drs. Warner, Tanaka, and Yada
for your mentorship and showing me what it means to be and how to be a mentor.
To my family, simply put I wouldn’t be here without you. I specifically want to thank you Kenny and
Jocelyn as the both of you have never, ever, shown anything but support and love along my BSc, MSc,
and throughout my PhD. You have both always freely offered your time, advice, support, and love in a
way that makes me feel blessed to be a part of and shown me what it truly means to be family. To Gayle
and Norm you have always both given so freely of yourselves along that way that I’ve truly felt more like
a son than a son-in-law. Getting to the end of this adventure and starting our next has made me more
thankful than ever to have you both in my life and appreciate how this success truly was a team effort.
To my best friend, my wife, and the mother of my child Kristina, we did it! It sounds like lip service to
say that this is your success as much of mine because it doesn’t cover all the ups and the downs, early
mornings, late nights, doubts, moving across the country…twice, and success along the way. You had the
strength and wisdom to be my biggest supporter and my strongest critic when I needed it and not
necessarily when I wanted it and I couldn’t have done it without you my love. I love you Kristina and
can’t wait to start our next adventures together!

v
Table of Contents
Abstract .................................................................................................................................... i
Acknowledgements .................................................................................................................. iii
Table of Contents ..................................................................................................................... iv
List of Tables ........................................................................................................................... x
List of Figures .......................................................................................................................... xii
List of Abbreviations ............................................................................................................... xvi
List of Equations ...................................................................................................................... xviii
Chapter 1 – INTRODUCTION .............................................................................................. 1
1.1 Literature Review ..................................................................................................... 1
1.1.1 Role of Industrial Enzymes ................................................................................... 1
1.1.2 Industrial Enzymatic Design ................................................................................. 2
1.1.3 Rationale Enzymatic Design ................................................................................. 4
1.1.4 Bioinformatic Tools............................................................................................... 6
1.1.5 Directed Evolution Enzyme Design ...................................................................... 9
1.1.6 Proteolytic Enzymes .............................................................................................. 11
1.1.7 Aspartic Proteinase – An Overview ...................................................................... 13
1.1.8 Aspartic Proteinase – Renin .................................................................................. 18
1.2 Research Question ......................................................................................................... 20
1.3 Objectives ...................................................................................................................... 20
1.5 References ..................................................................................................................... 21
vi
Chapter 2 – EXPLORING THE ALKALINE INSTABILITY OF PEPSIN THROUGH
RATIONALE ENGINEERING............................................................................................... 31
2.1 Abstract ......................................................................................................................... 31
2.2 Introduction ................................................................................................................... 32
2.3 Materials and Methods .................................................................................................. 34
2.3.1 Generation of Pepsin Mutants ............................................................................... 34
2.3.2 Bioinformatic Tools............................................................................................... 34
2.3.3 Protein Expression, Purification & Activation of Mutant and Wild-Type Pepsin 35
2.3.4 Kinetic Assay......................................................................................................... 36
2.3.5 CD Spectroscopy ................................................................................................... 36
2.3.6 Kinetic Stability of Pepsin ..................................................................................... 37
2.4 Results ........................................................................................................................... 38
2.4.1 Bioinformatic Analysis .......................................................................................... 38
2.4.2 Kinetic Parameters................................................................................................. 42
2.4.3 CD Spectroscopy ................................................................................................... 52
2.4.4 Kinetic Stability ..................................................................................................... 53
2.5 Discussion ..................................................................................................................... 62
2.5.1 Bioinformatic Results ............................................................................................ 62
2.5.2 Proteolytic Activity ............................................................................................... 63
2.5.3 Secondary Structure ............................................................................................... 69
2.5.4 Kinetic Stability ..................................................................................................... 71
2.6 Conclusion ..................................................................................................................... 73
2.7 Acknowledgements ....................................................................................................... 74
vii
2.8 Supplemental Material .................................................................................................. 75
2.9 References ..................................................................................................................... 78
Chapter 3 – A COMPARATIVE BIOINFORMATIC ANALYSIS OF PEPSIN AND RENIN
........................................................................................................................................ 84
3.1 Abstract ......................................................................................................................... 84
3.2 Introduction ................................................................................................................... 84
3.3 Materials & Methods ..................................................................................................... 88
3.3.1 Superpose .............................................................................................................. 88
3.3.2 UCSF Chimera ...................................................................................................... 88
3.3.3 PRALINE .............................................................................................................. 88
3.3.4 Zebra ...................................................................................................................... 89
3.3.5 Consurf .................................................................................................................. 89
3.3.6 Globplot ................................................................................................................. 89
3.3.7 Solvent Accessible Surface Area........................................................................... 89
3.3.8 Translation Liberation Screw Motion Determination (TLMSD) .......................... 90
3.3.9 CABS-flex protein flexibility ................................................................................ 90
3.3.10 CNAnalysis.......................................................................................................... 90
3.3.11 Protein Structure Visualization ............................................................................ 90
3.4 Results and Discussion .................................................................................................. 91
3.4.1 Sequence and Structure Similarity ........................................................................ 91
3.4.2 Zebra: Bioinformatic Analysis to Identify Residues Responsible for Functional
Diversity ......................................................................................................................... 95
3.4.3 Protein Disorder Profile ......................................................................................... 99
viii
3.4.4 Amino Acid Composition Optimisation for Environment .................................... 102
3.4.5 Importance of Glycosylation to Alkaline Stability ................................................ 103
3.4.6 Solvent Accessible Surface Area Differences ....................................................... 106
3.4.7 TLMSD Differences between Pepsin and Renin ................................................... 107
3.4.8 Protein Residue Fluctuation determined by CABS-Flex ...................................... 109
3.4.9 Simulated Protein Denaturation and Thermostability by CNAnalysis .................. 110
3.5 Conclusion ..................................................................................................................... 119
3.6 References ..................................................................................................................... 120
3.7 Supplemental Materials ................................................................................................. 129
Chapter 4 – CONCLUDING REMARKS AND FUTURE WORK ...................................... 150
4.1 General Conclusions ..................................................................................................... 150
4.2 Future Directions ........................................................................................................... 151
4.3 References ..................................................................................................................... 153
Appendix A – CONCLUDING REMARKS AND FUTURE WORK ................................... 154
A.1 References .................................................................................................................... 165

ix
List of Tables
Table 2.1: Stabilization centers identified by SRide in the N-terminal lobe of pepsin ........... 40
Table 2.2: Stabilization centers identified by SRide in the C-terminal lobe of pepsin ............ 41
Table 2.3: Stabilization centers identified by SRide that differ in sequence to that of renin .. 43
Table 2.4: Aspartic and glutamic residues identified as abnormally protonated by H++ ....... 44
Table 2.5: Residues identified through literature or via bioinformatic analysis thought to play a
role in stability ......................................................................................................................... 45
Table 2.6: k and K kinetic values for mutants across the pH range tested by nonlinear
cat m
regression using the method of least-squares for best fit to the Michaelis-Menten model...... 46
Table 2.7: k /K kinetic values for mutants across the pH range tested by nonlinear regression
cat m
using the method of least-squares for best fit to the Michaelis-Menten model ....................... 47
Table 2.8: Change in α-, β-, turn, and unordered structure compared to WT at pH 5.5 .......... 54
Table 2.9 Difference in β-sheet total content vs. WT at pH 5.5 .............................................. 55
Table 2.10: Ln(k ) values determined via unfolding experiments for mutants of interest .... 60
unf
Table 2.11: Dependence of free energy of unfolding on denaturant concentration determined via
unfolding experiments values for mutants of interest .............................................................. 61
Table 2.A: Primers utilized to generate pepsin mutants .......................................................... 75
Table 3.1: Superpose superposition of enzymes showing evolutionary relevance to renin with
pepsin (4PEP) as the reference structure for comparison. ....................................................... 92
Table 3.2: Subfamily-specific positions identified by Zebra analysis in pepsin-renin select
sequences ................................................................................................................................. 100
Table 3.A Subfamily-specific positions identified by Zebra in pepsin select sequences ........ 134
x