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Investigation of biological macromolecules using atomic force microscope-based techniques [Elektronische Ressource] / von Christian A. Bippes

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165 pages
    Investigation of Biological Macromolecules Using Atomic Force Microscope‐Based Techniques  Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften an der Technischen Universität Dresden von Christian A. Bippes geboren am 19.11.1978 in Karlsruhe Datum der Einreichung: 18.05.2009 1. Gutachter: Prof. Dr. Daniel J. Müller 2. Gutachter: Prof. Dr. Dimitrios Fotiadis Datum der Disputation: 18.08.2009     Meinen Eltern.    SUMMARY The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example.
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InvestigationofBiologicalMacromoleculesUsingAtomicForceMicroscopeBasedTechniques
Dissertation
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.) vorgelegt der
Fakultät Mathematik und Naturwissenschaften
an der Technischen Universität Dresden von
ChristianA.Bippes
geboren am 19.11.1978 in Karlsruhe Datum der Einreichung: 18.05.2009 1. Gutachter: Prof. Dr. Daniel J. Müller 2. Gutachter: Prof. Dr. Dimitrios Fotiadis Datum der Disputation: 18.08.2009
Meinen Eltern.
SUMMARY
The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques.
In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies.
A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods.
A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed.
Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT fromBacillussubtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural
segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane.
Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.
TABLEOFCONTENTS
TableofContents
C 1HAPTERINTEGRALMEMBRANEPROTEINS..................................................................................1 1.1 CELLULARMEMBRANES........................................................................................... 1 1.2 MEMBRANEPROTEINS............................................................................................. 3 1.2.1 OntheImportanceofMembraneProteinFoldingandAssembly....................... 5 1.2.1.1 ProgressinProteinFolding ................................................................................... 5 1.2.1.2 MembraneProteinFolding .................................................................................... 7 TransloconAssistedMembraneProteinFoldingandInsertion......................... 7 TheʺTwoStageʺModel ..................................................................................... 8 TheʺFourStepʺModel ...................................................................................... 9 1.2.1.3 MembraneProteinMisfolding ............................................................................. 11 1.2.2 HurdlesinMembraneProtein12Studies ................................................................... 1.2.3 MethodologicalApproachesinMembraneProteinResearch ............................ 13 1.3 MEMBRANEPROTEINSFULLFILSPECIFICFUNCTIONS....................................... 15 1.3.1 GapJunctions............................................................................................................... 15 1.3.2 AminoAcidTransporters .......................................................................................... 18 1.3.2.1 HowCellsPerformTransportAcrossMembranes .............................................. 18 1.3.2.2 LAminoAcidTransport...................................................................................... 19 C 2HAPTERA F M ......................................................................................21 TOMICORCEICROSCOPY 2.1 HISTORY................................................................................................................... 21 2.2 PRINCIPLE................................................................................................................ 21 2.2.1 AFMSetup.................................................................................................................... 21 2.2.2 Cantilevers .................................................................................................................... 23 2.2.2.1 GeneralConsiderations ........................................................................................ 23 2.2.2.2 ForcesActingontheCantilever ........................................................................... 24 2.2.2.3 ForceSensitivity................................................................................................... 26 2.2.2.4 CantileverCalibration .......................................................................................... 27 DetectorCalibration.......................................................................................... 27 SpringConstantCalibrationAddedMass..................................................... 28 SpringConstantCalibrationSaderMethod .................................................. 29 SpringConstantCalibrationThermalFluctuationAnalysis ........................ 29 2.3 OPERATIONMODES............................................................................................... 30 2.3.1 SurfaceImaging........................................................................................................... 31 2.3.1.1 ContactMode ....................................................................................................... 31 2.3.1.2 Tapping32Mode ...................................................................................................... 2.3.2 ForceMeasurement ..................................................................................................... 33 2.3.2.1 AFMBasedSingleMoleculeForceSpectroscopy ................................................ 35
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ConstantVelocityForceSpectroscopy.............................................................. 35 ConstantForceForceSpectroscopy .................................................................. 36 2.3.2.2 PolymerExtensionModels .................................................................................. 36 FreelyJointedChainModel .............................................................................. 37 WormlikeChainModel .................................................................................... 37 2.3.2.3 Model38Systems ..................................................................................................... Dextran............................................................................................................. 39 th 27ImmunoglobulinDomainfromtheGiantMuscleProteinTitin............... 40 Bacteriorhodopsin ............................................................................................. 41 2.3.2.4 KineticInterpretationofSMFS41Experiments ...................................................... 2.4 WHYUSEATOMICFORCEMICROSCOPYTOSTUDYMEMBRANEPROTEINS?. 44 2.4.1 ImagingMembraneProteinSurfacesatHighResolution .................................. 44 2.4.2 ForceasanAlternativeDenaturant ......................................................................... 44 CHAPTER3VE S D M U B ISCO LASTICITYOFINGLEEXTRANOLECULESNRAVELEDBYROWNIANNOISEANALYSIS........................................................................................................... 47 3.1 INTRODUCTION....................................................................................................... 47 3.2 EXPERIMENTALPROCEDURES................................................................................ 48 3.2.1 SamplePreparation .................................................................................................... 48 3.2.2 AFMInstrumentation ................................................................................................ 48 3.2.3 ExtractingViscoElasticProperties .......................................................................... 49 3.3 RESULTSANDDISCUSSION.................................................................................... 50 3.3.1 ExtractionofViscoElasticParameters.................................................................... 50 3.3.2 MeasurementNoiseDependsonWindowSizeandCantileverType ............. 51 3.3.3 ViscoElasticityofDextran........................................................................................ 54 3.4 CONCLUSIONS......................................................................................................... 56 C 4HAPTERD FF SM FC S IGITALORCE EEDBACKFORINGLE OLECULEORCE LAMPPECTROSCOPYNPROTEINS................................................................................................................. 57 O 4.1 INTRODUCTION....................................................................................................... 57 4.2 EXPERIMENTALPROCEDURES................................................................................ 58 4.2.1 SupportPreparation ................................................................................................... 58 4.2.2 SamplePreparation .................................................................................................... 59 4.2.3 ForceSpectroscopyMeasurements.......................................................................... 59 4.2.4 DataAnalysis............................................................................................................... 61 4.2.5 SoftwareBasedPI(D)Controller ............................................................................. 61 4.3 RESULTSANDDISCUSSION.................................................................................... 62 4.3.1 PI(D)Controller .......................................................................................................... 62 4.3.2 SystemStability .......................................................................................................... 64 54.3.3 ForceClampUnfoldingof65Ig27 .............................................................................. ii
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4.3.4 ForceClampUnfoldingofNhaA ............................................................................. 65 4.4 CONCLUSIONS......................................................................................................... 68 CHAPTER5L B T E L M P IGANDINDINGRIGGERSNERGYANDSCAPEANDECHANICALROPERTIESA A A S T ....................................................................69 OFTHEMINOCIDNTIPORTERTE 5.1 INTRODUCTION....................................................................................................... 69 5.2 EXPERIMENTALPROCEDURES................................................................................ 71 5.2.1 Cloning,Overexpression,Purification,andReconstitutionintoProteoliposomesofSteT............................................................................................................................ 71 5.2.2 SMFSandDFS ............................................................................................................. 71 5.2.3 DataSelectionandAnalysis...................................................................................... 72 5.2.4 CompensationforHydrodynamicDrag.................................................................. 73 u05.2.5 CalculationofxandkfromDFSData .................................................................. 74 5.2.6 CalculationofTransitionBarrierHeightsand74Rigidity ...................................... 5.3 RESULTS................................................................................................................... 75 5.3.1 InteractionsofSteTInPresenceandAbsenceofSubstrates .............................. 75 5.3.2 DirectionofUnfolding............................................................................................... 78 5.3.3 ProbabilityofInteractions......................................................................................... 78 5.3.4 SubstrateBindingChangestheDynamicEnergyLandscapeofSteT............... 79 u05.3.5 Correlationofx,k,andΔG..................................................................................... 83 5.3.6 MechanicalPropertiesofSteT .................................................................................. 84 5.4 DISCUSSION............................................................................................................ 85 5.4.1 AminoAcidBindingbySteTLacksDetectableLocalizedInteractions ........... 85 5.4.2 SteTUnfoldsDifferentlyComparedtoOtherMembraneProteins .................. 86 5.4.3 SubstrateBindingChangestheEnergyLandscapeofSteT ................................ 88 5.4.4 HammondLikeBehaviorReflectsGroundStateEffects .................................... 89 5.5 CONCLUSIONS......................................................................................................... 90 CHAPTER6PHINDUCEDCONFORMATIONALCHANGESINCX26HEMICHANNELSM A ..........................................................................93 ODULYTEDBYMINOSULFONATES 6.1 INTRODUCTION....................................................................................................... 93 6.2 EXPERIMENTALPROCEDURES................................................................................ 95 6.2.1 Cx26GapJunctionPreparation................................................................................. 95 6.2.2 AFMImaging ............................................................................................................... 95 6.2.3 ImageProcessingandAveraging ............................................................................. 96 6.3 RESULTS................................................................................................................... 96 6.3.1 HighResolutionImagingoftheExtracellularConnexonSurface..................... 96 6.3.2 ObservingpHInducedConformationalChanges ................................................ 98 6.3.3 ConformationalChangesAreNotDependentonHEPESConcentration ...... 101
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6.3.4 NoConformationalChangeOccursintheAbsenceofAminosulfonates ..... 102 6.3.5 QuantitativeAnalysisoftheChannelClosure ................................................... 103 6.4 DISCUSSION........................................................................................................... 104 6.4.1 AminosulfonatesAreRequiredtoInduceClosureDuringAcidificationinIsolatedCx26GapJunctionHemichannels ......................................................... 105 6.4.2 pHGatingofConnexins.......................................................................................... 106 6.4.3 MechanismofChannelClosureattheExtracellularSurfaceGate.................. 107 6.4.4 ConnexonExtracellularSurfaceIsMoreRigidWhentheExtracellularGateIsClosed.......................................................................................................................... 109 6.4.5 TheRelevanceofpHGatinginTissuesandOrgans ......................................... 109 6.4.6 TheImportanceofTaurineinTissuesandOrgansandCoExpressionwithCx26 110 6.5 CONCLUSIONS....................................................................................................... 111 C 7HAPTEROUTLOOK.................................................................................................................... 113 7.1 EFFECTOFLIGANDSANDINHIBITORSONTRANSPORTPROTEINS................ 114 7.2 MEMBRANEPROTEINFOLDING.......................................................................... 114 CHAPTER8APPENDIX.................................................................................................................... 117 GLOSSARYOFABBREVIATIONS.................................................................................... 117 GLOSSARYOFSYMBOLS................................................................................................. 120 PUBLICATIONLIST.......................................................................................................... 123 ACKNOWLEDGEMENTS.................................................................................................. 125 CHAPTER9BIBLIOGRAPHY............................................................................................................ 127
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Chapter1:IntegralMembraneProteins
INTEGRALMEMBRANEPROTEINS
1.1 CELLULARMEMBRANES
Chapter 1
The cell is the building block of all organisms. In its simplest form, a cell can be described as a balloon filled with various macromolecules and chemicals that are essential for life, e.g. proteins, nucleic acids, carbohydrates, and small organic and inorganic molecules [1]. That balloon is called the outer or plasma membrane and is universal to all cells on earth.
In the early prebiotic environment, various simple organic molecules formed, which then reacted to form more and more complex molecules. In the course of time, 1 intricate catalytic and self-replicating polymeric systems developed . While these systems coexisted, they also had to compete for available resources in the primordial pond. The development of the plasma membrane was the advent of the first cell-like structures and a milestone in evolution [1]. Improvements in the "cellular" machinery, which proved beneficial in the struggle for survival, did not have to be shared with competing, free-floating systems. Nutrients could be gathered from the environment and products of the cell's metabolic machinery could be retained within the cell. Thus, the plasma membrane promoted further growth and evolution of the cell. Even "simple" prokaryotes, containing only a simple plasma membrane, which is often surrounded by a cell wall, performs various metabolic tasks in different regions of the cytoplasm. In more complex eukaryotes, membranes define different organelles, such as endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplasts, lysosomes, peroxisomes, or vacuoles [1]. In all cases compartmentalization preserves the (bio)chemical and physical characteristics of organelles.
Most probably, spontaneous self-assembly of lipids present in the primordial pond lead to formation of the first plasma membrane [2]. However, it is intrinsic to 1  The first self-replicating systems are believed to be ribonucleic acids. Although they show limited catalytic activities compared to proteins, they can easily provide for exact (complementary) copies of themselves [2]. 1
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