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«THE FUNCTIONAL INCORPORATION OF THE MECHANOSENSITIVE CHANNEL OF LARGE CONDUCTANCE WITHIN A TETHERED LIPID BILAYER AND THE FUTURE RECONSTITUTION OF ...»

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THE FUNCTIONAL INCORPORATION OF THE MECHANOSENSITIVE CHANNEL OF

LARGE CONDUCTANCE WITHIN A TETHERED LIPID BILAYER AND THE FUTURE

RECONSTITUTION OF FUTURE DESIGNER CHANNELS

By

DANYELL S. WILSON

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

© 2009 Danyell Wilson This document is dedicated to my grandmother, Ms. Addie Martin.

ACKNOWLEDGMENTS

First I would like to thank God for giving me the strength to complete this document. I would also like to thank my committee members, Dr. Randolph Duran, Dr. Paul Blount, Dr. Gail Fanucci, Dr. Joanna Long, Dr. Benjamin Smith, and Dr Tom Lyons, for their advice, guidance, and support. Dr. Blount was a second advisor and a mentor to me; sincere thanks for opening up the Blount’s lab to me. Dr. Fanucci and Dr. Lyons special thanks for opening up the biochemistry labs at UF to me as well. These facilities allowed me perform and complete all of the biochemistry techniques I learned while in Dr. Blount’s lab. Dr. Duran, Dr. Smith and Dr.

Long, a special thanks for encouraging me to continue on to the PhD after my Masters.

I would next like to thank Dr. Martin Andersson, Dr. Irene Iscla, Dr. Li-Min Yang, Senior Scientist (Dr.) Robin Wray, and soon to be Dr. Mandy Blackburn as well as Chenyu Zhu, Jeanette Cervantes, Deneyelle Wilson, and Alan Salgado. I would also like to thank the Duran group members for always being there.

Next, I would love to thank my family and friends. My mother Marjorie Wilson who always, always has my back; I am truly grateful to have such a loving charismatic mother. I would love to thank my father, Danny Wilson; a strong, smart, funny, and a blessing to have as a father and friend. I would like to also thank my sisters Sunceray and Ariel Wilson; I am truly blessed to have both of my sisters in life. Sunceray and Ariel were always just a phone call away and for that I will always be sincerely grateful. I would like to thank my nephew, Jameź Epps, as my inspiration, and the best nephew, ever. I would like to thank all of my friends from childhood, college, graduate school, and church. Without them, I would not have made it through this process. All of their prayers, laughter, consoling, partying, editing, and the staying up late nights with me to ensured I became Dr. Wilson. I would also like to acknowledge my dear friend Dr. Charlee Bennett; we did it! We are Ph officially Done!

Lastly, I would like to thank the people who have financially supported me and helped me find my passion in science: Dr. Anna Donnelly (SEAGEP), Dr. Jonathan Earl and Ms. Margie Williams (FGLAMP), the Office of Graduate Minority Programs (BOE), and both Dr. Blount and Dr. Duran.

–  –  –

ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

ABSTRACT

CHAPTER 1 PROJECT OVERVIEW

Introduction

Biological Membrane

Membrane Proteins and Ion Channels

Stretch-Activated Channels

E. coli’s MscL

MscL Function and Structure

S3-COOH-Bundle

Functionality of the S3-Bundle

Solid-Supported and Tethered Lipid Bilayers

Tethered Lipid Bilayers/Membranes (tBLM)

DPTL

Surface Binding Region

The Polar Tethered/Hydrophilic Region

Diphytanyl Tail Regions

Diphytanoyl Lipids

Summary

2 MATERIALS, METHODS, AND EXPERIMENTAL THEORIES

Protein Chemistry and Structural Studies

Cysteine Mutations in MscL

Biochemistry Assays: Disulfide-trapping

Western Blotting

Experimental Theory and Procedures for Functional Studies

Materials

Protein Isolation and Purification

MscL Isolation and Purification

Gramicidin Isolation

Proteoliposome Formation

DPhPC and DPhPE Vesicle Formation and Protein Reconstitution

Single Channel Measurements: Patch Clamping Recording Technique

Specialized Patch Clamping Techniques

Planar Linear Patching: The Tip-Dip Recording

Tethered Lipid Bilayer

Experimental Setup

Tip-Dip Recording Experiment

Tethered Devices Experimental Setup

The Device

Preparation of the Tethered Bilayer

Gigaseal

Program Analysis for Both Techniques

Analysis of Single Ion Channel Activity

Point-Amplitude Histograms

Probability Density Function

3 CYSTEINE SUBSTITUTIONS IN THE S3-BUNDLE OF THE

MECHANOSENSITIVE CHANNEL OF LARGE CONDUCTANCE

Introduction

Materials and Methods

Strains and Cell Growth

Phenotype studies

GOF Assays

Loss-of-Function Assays

Western Blot Analysis

Electrophysiology

Results

Phenotype Characterization

GOF Studies

LOF Assays

Biochemical Assays





Discussion

Conclusion

4 TESTING THE ELECTRICAL STABLITY OF DIFFERENT LIPID BILAYER

COMPOSITIONS OF DPhPC/DPhPE

Introduction

Methods and Materials

Results of Lipid Ratios

Pure PC Planar Bilayers

Pure PE Planar Bilayers

50PC/50PE Planar Bilayers

70PC/30PE and 30PC/70PE Planar Bilayers

Single Channel Results of Gramicidin in 70PC/30PE bilayer

Discussion

The Effect of Hydration State on the Stability of Bilayers and Electroporation...........108 Tip-dip and Diphytanoyl Lipid System

Conclusion

5 FUNCTIONAL STUDIES OF GRAMICIDIN AND MSCL WITHIN A TETHERED

LIPID BILAYER MEMBRANE

Introduction

Materials and methods

Materials

Proteins

Device

Preparation of the Tethered Bilayer

Protein Reconstitution

Electrophysiology

Results

Results of Gramicidin Reconstituted into the tBLM

Single Channel Analysis of MscL

Ionic Selectivity

Lifetime Studies

Conductance

Discussion

Conclusions

6 CONCLUSION

APPENDIX A DLS OF THE SIZE DISTRIBUTION OF 70PC/30 PE

B STABLE BILAYER TRACE AND I-V CURVES

C PROTEIN RECTIFICATION WITHIN THE DEVICE

LIST OF REFERENCES

BIOGRAPHICAL SKETCH

–  –  –

1-1 Different types of ion channels.

2-1 Oligonucleotide primers with cysteine mutation highlighted in red

5-1 Conductance comparison between the ion channels reconstituted in the tBLM and reported conductance.

–  –  –

1-1 Phospholipids bilayer. And The fluid mosaic model of the cellular membrane..............18 1-2 Equivalent circuit model of the ion channel within the lipid membrane.

1-3 Schematic of Gramicidin forming a pore in a synthetic lipid bilayer membrane..............23 1-4 Schematic representation of E. coli’s MscL amino acid sequence and topology of one monomer.

1-5 Helical wheel of the S3-bundle

1-6 Conflicting models of the S3-bundle during gating

1-7 Solid-Confined or supported membrane

1-8 The tethered monolayer component

1-9 1,2-Diphytanoyl-sn-Glycero-3-phosphoethanolamine (DPhPE) and 1,2-Diphytanoylsn-Glycero-3-phospho choline (DPhPC) lipid

2-1 Liposome or vesicles formed from phospholipids

2-2 Schematic of the tip-dip recording system

2-3 Experimental Apparatus

2-4 The tip-dip and tethered stage apparatus

2-5 Event detection occurring at level zero and level one

2-6 The distribution of data points within the histogram are fitted by the maximum likelihood with a continuous Gaussian curve..

3-1 Conflicting models of the S3-bundle during gating

3-2 Amino acid sequence for MscL and a ribbon design of the linker area and S3 bundle of the C-terminal region with the location of the mutations highlighted

3-3 Schematic illustration of gain of function assay.

3-4 Loss of Function schematic representation

3-5: Video images obtained during the process of generating and patching giant spheroplasts from E. coli

3-6 Growth curves for all of the mutants generated in this study as well as for WT MscL and G26H.

3-7 LOF study analyzing the percent survival of the mutants.

3-8 Percentage of Dimerization for single mutants A110C, A111C, A112C, and A114C.....85 3-9 Disulfide crosslinking in single and double mutants identified using western blotting..

3-10 Monomers of single mutants and WT as a control exposed to reducing agent DTT.........86 3-11 Double mutants E119C/V120C, L121C/L122C, L128C/L129C, and AAA exposed to

0.5 M NaCl-LB, water (2nd row), 0.5 M NaCl-LB and 1% H2O2 (3rd row), and water with 1% H2O2..

3-12 Single channel traces of A110H and A112H when exposed to regular patch clamping buffer, and a patch solution of ZnCl2

3-13 Closed and opened state of MscL with mutations generated in this study highlighted.....93 4-1 A reversible hole in the bilayer formed from electroporation with an applied potential of -138 mV, and a current transition of more then -200 pA.

4-2 Mini-electroporation of a 100% DPhPC bilayer at 80 mV. Pores have a uniform current flow of 10 pA

4-3 SEM images of the borosilicate pipette tips.

4-4 Electroporation of 100% PC at 120mV with a bilayer lifetime of 56 minutes................101 4-5 Electroporation of 100% PE planar lipid bilayer with potentials applied from 100mV-180 mV.

Electroporation occurring with a 50PC/50PE bilayer within the 1st 25 sec

4-6 4-7 Ramp voltages applied to the 70PC/30PE lipid mixture.

4-8 Episodic trace of the current in pA obtained lipid bilayer composed of 70PC/30PE......104 4-9 An 18 minute trace of 70PC/30PE lipid mixture.

4-10 Single channel conductance of Gramicidin A is around 67 pS when 60 mV is applied

4-11 Plots of a 1.5 second run at negative 100 mV with and without Gramicidin..................106 4-12 Plots IV curves for Gramicidin A for various KCl concentrations

5-1 Tethered bilayer membrane array.

5-2 AFM images of the monolayer deposition and vesicle fusion process

5-4 A stable tethered bilayer form from vesicle fusion of 70PC/30PE LUVs

5-5 Single channel activity of Gramicidin at an applied voltage of 60 mV with a conductance of 60 pS..

5-6 I-V curves of Gramicidin in the tBLM on the device and within the tip-dip system......125 5-7 Single channel activity of multiple Gramicidin channels opened within the tethered lipid bilayer.

5-8 Single channel activity of WT MscL in the tLBM system: When 300 mV was applied to the membrane MscL opened conducting 266 pS..

5-9 MscL displaying two conducting-states:

5-10 MscL with the lysine in the 31 position highlighted

5-10 Single channel recordings of K31E (voltage sensitive mutant of MscL) within the tBLM

5-11 Single channel activity of K31E at 143 mV. ions was 60 pA providing a conductance of 350 pS

–  –  –

DPhPC 1,2-diphytanoyl-sn-glycero-3-phosphocholine DPhPE 1,2-diphytanoyl-sn-3-phosphoethanolamine DPTL 2,3-di-O-phytanoyl-sn-glucero-1-tetraethylene glycerol-D,L-α-lipoic acid ester lipid GOF Gain of Function LOF Loss of Function MscL Mechanosensitive Channel of Large Conductance tBLM Tethered Bilayer lipid Membrane

–  –  –

When creating a biosensor based on single ion channel activity, the conformational changes within a protein as well as the protein’s ability to reconstitute successfully into nonnative lipid environment and remain active must be understood. Therefore, the proximity of key residues in the C-terminal region of the Mechanosensitive Channel of Large Conductance (MscL) was quantified via disulfide bridging of cysteine mutations made to conserved hydrophobic residues in the linker and S3-bundle of this region. The biochemical assays utilizing disulfide bridging provided insight into which residues were dynamic and interactive, enabling them to be studied further via patch clamping. The channel’s C-terminal region during gating was under debate and our results support the theory of the S3-bundle remaining closed during gating. Results from this study also enabled the generation of two new mutant channels that could coordinate heavy metals and the recognition response was a decrease in conductance, and slowed channel kinetics.

After the correct configuration of the protein was established, the ideal lipid environment for studying single ion channels in a tethered device for biosensor applications was investigated.

The tip-dip electrophysiology method was used to determine an electrically stable lipid environment between different diphytanoyl compositions. Diphytanoyl lipids were vital components of this research due to their increased durability and stability when tethering to solid supports such as the gold surface on the device. Results indicated all of the lipid-ratios suffered from pore formation due to electrical breakdown, both reversible and irreversible. This was the first time that electroporation was reported at such low potentials as 125 mV and 40 mV which may be characteristic of using the tip-dip method with diphtanoyl lipids. The pore formation was random; however distinguishable from single ion channel conductance by configuring I-V curves and evaluating the kinetics of the pores formed in comparison to channel activity. The lipid ratio of 70PC/30PE was chosen as the most stable lipid ratio and was integrated as the synthetic lipid environment for both Gramicidin and MscL on the tBLM device. Results for Gramicidin, indicated that single channel activity within the tBLM were characteristic of the channel. On the other hand, a high applied voltage was required to gate MscL and the conductance response was lower for this channel in comparison to when it is in its natural environment. This lead to the incorporation of a voltage sensitive MscL mutant, K31E, which sensed tension when voltages as low as 85 mV were applied. Preliminary results indicate that this mutant is very active within the tBLM and single channel activity is attainable.



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