Biophysical chemistry of proteins an introduction to laboratory methods

As you can see from thefigure, light from each point of the light source is spread evenly across the entireobject planes, passing the object in parallel beams from every azimuth.. 1.1 Si

Biophysical Chemistry of Proteins

Ross University School of Medicine

Springer New York Dordrecht Heidelberg London

© Springer Science+Business Media, LLC 2011

All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,

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or by similar or dissimilar methodology now known or hereafter developed is forbidden.

The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject

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Printed on acid-free paper

Springer is part of Springer Science+Business Media ( www.springer.com )

During undergraduate courses in biochemistry you learned what proteins do as

enzymes, receptors, hormones, motors or structural components The more

inter-esting question, how proteins can achieve all these functions, is usually asked only

in graduate courses, and in many cases it is a topic of ongoing research

Here I want to present an overview of the methods used in such research projects,their possible applications, and their limitations I have limited the presentation to

a level where a general background in chemistry, physics, and mathematics is cient to follow the discussion Quantum mechanics, where required, is treated in apurely qualitative manner A good understanding of protein structure and enzymol-ogy is required, but these topics I have covered in a separate volume [44]

suffi-Apart from graduate training in protein science this book should also be useful

as a reference for people who work with proteins

After studying this book you should be able to collaborate with workers who havethe required instruments and use these methods routinely You should also be able

to understand papers which make use of such methods However, before embarking

on independent research using these methods you are directed to the literature citedfor a more in-depth, more quantitative coverage

This book focuses on the biophysical chemistry of proteins The use of nucleicacid-based methods [360], although in many cases very relevant and informative, isoutside the scope of this text Also only hinted at are modern approaches to com-putational biochemistry [20, 180, 231] In the end, the models derived from suchtechniques have to be verified by experiments If this book stimulates such studies,

it has served its purpose

Acknowledgements

I wish to thank all my students, friends, and colleagues who have given me theirsupport and suggestions for this text, and who have gone through the arduoustask of proof-reading All remaining errors are, of course, mine Please reportany errors found and any suggestions for improvement to me (mailto://engelbertbuxbaum@web.de)

v

A big “thank you” goes to all those who have made software freely available, orwho maintain repositories of information on the internet Without your dedication,this book would not have been possible.

Part I Analytical Techniques

1 Microscopy 3

1.1 Optical Foundations of Microscopy 3

1.1.1 KOHLER-Illumination 3

1.1.2 The Role of Diffraction 5

1.1.3 The Importance of the Numerical ApertureNa 7

1.1.4 Homogeneous Immersion 9

1.1.5 Lens Aberrations 10

1.1.6 Special Methods in Light Microscopy 12

1.2 The Electron Microscope 17

1.2.1 Transmission Electron Microscopy 17

1.2.2 Scanning Electron Microscopy 20

1.2.3 Freeze Fracture 20

1.3 Other Types of Microscopes 20

1.3.1 The Atomic Force Microscope 21

1.3.2 The Scanning Tunnelling Microscope 21

1.3.3 The Scanning Near-Field Optical Microscope 22

2 Single Molecule Techniques 23

2.1 Laser Tweezers and Optical Trapping 23

3 Preparation of Cells and Tissues for Microscopy 25

3.1 Fixing 25

3.2 Embedding and Cutting 26

3.3 Staining 26

3.4 Laser Precision Catapulting 26

4 Principles of Optical Spectroscopy 27

4.1 Resonant Interaction of Molecules and Light 27

4.2 The Evanescent Wave 29

vii

5 Photometry 33

5.1 Instrumentation 33

5.2 LAMBERT–BEER’s Law 33

5.2.1 The Isosbestic Point 36

5.3 Environmental Effects on a Spectrum 36

6 Fluorimetry 39

6.1 Fluorescent Proteins 40

6.2 Lanthanoid Chelates 41

6.2.1 Quantum Dots 44

6.3 Fluorescence Quenching 44

6.3.1 Dynamic Quenching 44

6.3.2 Static Quenching 46

6.4 F ¨ORSTERResonance Energy Transfer 46

6.4.1 Handling Channel Spillover 48

6.4.2 Homogeneous FRET Assays 49

6.4.3 Problems to Be Aware Of 49

6.4.4 Fluorescence Complementation 50

6.4.5 Pulsed Excitation with Multiple Wavelengths 50

6.5 Photoinduced Electron Transfer 50

6.6 Fluorescence Polarisation 52

6.6.1 Static Fluorescence Polarisation 53

6.6.2 Application 53

6.7 Time-Resolved Fluorescence 54

6.7.1 Fluorescence Autocorrelation 54

6.7.2 Dynamic Fluorescence Polarisation 55

6.8 Photo-bleaching 55

7 Chemiluminescence 57

7.1 Chemiluminescent Compounds 57

7.2 Assay Conditions 59

7.3 Electrochemiluminescence 59

8 Electrophoresis 61

8.1 Movement of Poly-ions in an Electrical Field 62

8.1.1 Influence of Running Conditions 62

8.2 Electrophoretic Techniques 66

8.2.1 Techniques of Historic Interest 67

8.2.2 Gel Electrophoresis 69

8.2.3 Free-Flow Electrophoresis 72

8.2.4 Native Electrophoresis 73

8.2.5 Denaturing Electrophoresis 77

8.2.6 Blue Native PAGE 78

8.2.7 CTAB-Electrophoresis 79

8.2.8 Practical Hints 79

Contents ix

8.2.9 IEF and 2D-electrophoresis 81

8.2.10 Elution of Proteins from Electrophoretic Gels 90

8.2.11 Gel Staining Procedures 90

8.2.12 Capillary Electrophoresis 94

9 Immunological Methods 97

9.1 Production of Antibodies 97

9.1.1 Isolation from Animals 97

9.1.2 Monoclonal Antibodies 100

9.1.3 Artificial Antibodies 101

9.1.4 Aptamers .102

9.2 Immunodiffusion 103

9.3 Immunoelectrophoretic Methods 104

9.4 RIA, ELISA and Immuno-PCR 104

9.4.1 RIA 105

9.4.2 ELISA 105

9.4.3 Immuno-PCR 107

9.5 Methods that Do Not Require Separation of Bound and Unbound Antigen .107

9.5.1 Microwave and Surface Plasmon Enhanced Techniques 110

9.6 Blotting 110

9.6.1 Western Blots 111

9.6.2 Dot Blots 114

9.6.3 Total Protein Staining of Blots 114

9.6.4 Immunostaining of Blots 115

9.7 Immunoprecipitation 117

9.8 Immunomicroscopy 117

9.9 Fluorescent Cell Sorting 119

9.10 Protein Array Technology 120

10 Isotope Techniques 123

10.1 Radioisotopes .123

10.1.1 The Nature of Radioactivity .124

10.1.2 Measuring“-Radiation .126

10.1.3 Measuring”-Radiation 131

10.2 Stable Isotopes 131

Part II Purification of Proteins 11 Homogenisation and Fractionisation of Cells and Tissues 135

11.1 Protease Inhibitors 136

12 Isolation of Organelles 141

13 Precipitation Methods 143

13.1 Salts 143

13.2 Organic Solvents 145

13.3 Heat 146

14 Chromatography 147

14.1 Chromatographic Methods .147

14.2 Theory of Chromatography .152

14.2.1 The CRAIG-Distribution 152

14.2.2 Characterising Matrix–Solute Interaction 155

14.2.3 The Performance of Chromatographic Columns 157

14.3 Strategic Considerations in Protein Purification 161

14.3.1 Example: Purification of Nucleotide-free Hsc70 From Mung Bean Seeds 161

15 Membrane Proteins 163

15.1 Structure of Lipid/Water Systems 163

15.2 Physicochemistry of Detergents 166

15.2.1 Detergent Partitioning into Biological Membranes 171

15.3 Detergents in Membrane Protein Isolation .174

15.3.1 Functional Solubilisation of Proteins 174

15.3.2 Isolation of Solubilised Proteins 176

15.3.3 Reconstitution of Proteins into Model Membranes 177

15.4 Developing a Solubilisation Protocol 179

15.5 Membrane Lipids: Preparation, Analysis and Handling .181

15.5.1 Measurements with Lipids and Membranes .181

16 Determination of Protein Concentration 183

17 Cell Culture 187

17.1 Cell Types 188

17.1.1 Contamination of Cell Cultures 189

Part III Protein Modification and Inactivation 18 General Technical Remarks 193

18.1 Determining the Specificity of Labelling 194

18.2 Kinetics of Enzyme Modification 194

19 Amine-Reactive Reagents 199

20 Thiol and Disulphide Reactive Reagents 205

20.1 Cystine Reduction .207

Contents xi

21 Reagents for Other Groups 209

21.1 The Alcoholic OH-Group .209

21.2 The Phenolic OH-Group 210

21.3 Carboxylic Acids 212

21.4 Histidine 213

21.4.1 Tryptophan .213

21.4.2 Arginine .215

21.4.3 Methionine .215

22 Cross-linkers 219

22.1 Reversible Cross-linkers 219

22.2 Trifunctional Reagents 220

23 Detection Methods 223

23.1 Radio-labelling of Proteins 223

23.2 Photo-reactive Probes 224

23.3 Biotin 224

23.4 Particle Based Methods 226

23.4.1 Colloidal Gold 226

23.4.2 Magnetic Separation .227

24 Spontaneous Reactions in Proteins 229

24.1 Reactions 229

24.1.1 Racemisation 229

24.1.2 Oxidation 230

24.1.3 Amyloid-Formation 230

24.2 Applications 232

Part IV Protein Size and Shape 25 Centrifugation 237

25.1 Theory of Centrifugation 238

25.1.1 Spherical Particles 238

25.1.2 Non-spherical Particles 240

25.1.3 Determination of Molecular Mass 241

25.1.4 Pelleting Efficiency of a Rotor 244

25.2 Centrifugation Techniques .245

25.3 Rotor-Types .246

25.4 Types of Centrifuges 247

25.5 Determination of the Partial Specific Volume .248

26 Osmotic Pressure 251

26.1 Dialysis of Charged Species: The DONNAN-Potential 252

27 Diffusion 255

28 Viscosity 257

29 Non-resonant Interactions with Electromagnetic Waves .261

29.1 Laser Light Scattering .261

29.1.1 Static Light Scattering 261

29.1.2 Dynamic Light Scattering .263

29.1.3 Quasi-elastic Scattering 264

29.1.4 Instrumentation 265

29.2 Small Angle X-ray Scattering SAXS .265

29.3 Neutron Scattering 265

29.4 Radiation Inactivation .266

Part V Protein Structure 30 Protein Sequencing 271

30.1 Edman Degradation 271

30.1.1 Problems that May Be Encountered 272

30.1.2 Sequxencing in the Genomic Age 273

30.2 Mass Spectrometry .274

30.2.1 Ionisers .274

30.2.2 Analysers (See Fig 30.6) .277

30.2.3 Determination of Protein Molecular Mass by Mass Spectrometry .280

30.2.4 Tandem Mass Spectrometry .281

30.2.5 Protein Sequencing by Tandem MS 282

30.2.6 Digestion of Proteins 284

30.2.7 Ion–Ion Interactions 284

30.3 Special Uses of MS 286

30.3.1 Disease Markers 287

30.3.2 Shotgun Sequencing of Proteins 287

30.4 Characterising Post-translational Modifications 287

30.4.1 Ubiquitinated Proteins 287

30.4.2 Methylation, Acetylation and Oxidation 287

30.4.3 Glycoproteins 288

31 Synthesis of Peptides 289

32 Protein Secondary Structure 291

32.1 Circular Dichroism Spectroscopy 291

32.2 Infrared Spectroscopy .294

32.2.1 Attenuated Total Internal Reflection IR-Spectroscopy 296

32.2.2 Fourier-Transform IR-Spectroscopy .296

32.2.3 IR-Spectroscopy of Proteins 297

32.2.4 Measuring Electrical Fields in Enzymes: The STARK-effect 301

32.3 Raman-Spectroscopy .302

Contents xiii

33 Structure of Protein–Ligand Complexes 303

33.1 Electron-Spin Resonance 303

33.1.1 Factors to Be Aware Of .304

33.1.2 Natural ESR Probes with Single Electrons .305

33.1.3 Stable Free Radical Spin Probes 305

33.1.4 Hyperfine Splitting: ENDOR-Spectroscopy 307

33.1.5 ESR of Triplet States 307

33.2 X-ray Absorbtion Spectroscopy 307

33.2.1 Production of X-rays 307

33.2.2 Absorbtion of X-rays 308

34 3-D Structures 309

34.1 Nuclear Magnetic Resonance 309

34.1.1 Theory of 1-D NMR 309

34.1.2 BOLTZMANN-Distribution of Spins 310

34.1.3 Parameters Detected by 1-D NMR 312

34.1.4 NMR of Proteins, Multi-dimensional NMR .314

34.1.5 Solid State NMR .318

34.2 Computerised Structure Refinement 319

34.2.1 Energy Minimisation 319

34.2.2 Molecular Dynamics 320

34.2.3 Monte Carlo Simulations .320

34.2.4 Future Directions 321

34.3 X-ray Crystallography of Proteins .321

34.3.1 Crystallisation of Proteins 322

34.3.2 Sparse Matrix Approaches to Experimental Design: The TAGUCHI-method 330

34.3.3 X-Ray Structure Determination .331

34.3.4 Other Diffraction Techniques 339

34.4 Electron Microscopy of 2-D Crystals 341

35 Folding and Unfolding of Proteins 343

35.1 Inserting Proteins into a Membrane 343

35.2 Change of Environment .344

35.2.1 Standard Conditions for Experiments 346

35.3 The Chevron-Plot 346

35.3.1 Unfolding by Pulse Proteolysis and Western-Blot 347

35.3.2 Non-linear Chevron-Plots 348

35.3.3 Unfolding During Electrophoresis .348

35.3.4 Membrane Proteins 349

35.4 The Double-Jump Test 349

35.5 Hydrogen Exchange .349

35.6 Differential Scanning Calorimetry 350

35.7 The Protein Engineering Method 350

Part VI Enzyme Kinetics

36 Steady-State Kinetics 355

36.1 Assays of Enzyme Activity 356

36.1.1 The Coupled Spectrophotometric Assay of WARBURG .357

36.2 Environmental Influences on Enzymes .359

36.2.1 pH 359

36.2.2 Ionic Strength .359

36.2.3 Temperature .360

36.3 Synergistic and Antagonistic Interactions .361

36.3.1 Nomenclature 361

36.3.2 The Isobologram .361

36.3.3 Predicting the Effect for Combinations of Independently Acting Agents 362

36.4 Stereoselectivity .364

37 Leaving the Steady State: Analysis of Progress Curves 367

38 Reaction Velocities 369

38.1 Near Equilibrium Higher Order Reactions can be Treated as First Order 369

38.2 Continuous Flow 370

38.3 Quenched Flow 371

38.4 Stopped Flow 372

38.5 Flow Kinetics 372

38.6 Temperature and Pressure Jumps 372

38.7 Caged Compounds 374

38.8 Surface Plasmon Resonance 374

38.8.1 Theory of SPR 377

38.8.2 Practical Aspects .378

38.8.3 Surface Plasmon Coupled Fluorescence 379

38.8.4 Dual Polarisation Interferometry 380

38.9 Quartz Crystal Microbalance 380

39 Isotope Effects .383

40 Isotope Exchange 387

40.1 ADP/ATP Exchange 387

40.2 18O-Exchange 387

40.3 Positional Isotope Exchange .388

Part VII Protein–Ligand Interactions 40.3.1 Structural Aspects of Protein–Protein Interactions 389

41 General Conditions for Interpretable Results 391

Contents xv

42 Binding Equations 393

42.1 The LANGMUIR-Isotherm: A Single Substrate Binding to a Single Binding Site 393

42.2 Binding in the Presence of Inhibitors 394

42.2.1 Competitive Inhibition 394

42.2.2 Non-competitive Inhibition 395

42.3 Affinity Labelling 396

42.3.1 Differential Labelling 397

43 Methods to Measure Binding Equilibria 399

43.1 Dialysis 399

43.1.1 Equilibrium Dialysis 399

43.1.2 Continuous Dialysis 400

43.2 Ultrafiltration 400

43.3 Gel Chromatography .402

43.3.1 The Method of HUMMEL ANDDREYER .402

43.3.2 Spin Columns 402

43.4 Ultracentrifugation 403

43.4.1 The Method of DRAPER AND V HIPPEL .403

43.4.2 The Method of STEINBACH ANDSCHACHMAN 403

43.5 Patch-Clamping 404

43.6 Mass Spectrometry .405

43.7 Determination of the Number of Binding Sites: The Job-Plot 406

44 Temperature Effects on Binding Equilibrium and Reaction Rate 409

44.1 Activation Energy 409

44.2 Isothermal Titration Calorimetry 412

44.2.1 Photoacoustic Calorimetry 413

Part VIII Industrial Enzymology 45 Industrial Enzyme Use 417

45.1 Enzyme Denaturation 419

45.2 Calculation of the Required Amount of Enzyme 420

46 Immobilised Enzymes 421

46.1 Kinetic Properties of Immobilised Enzymes 422

46.1.1 Factors Affecting the Activity of an Immobilised Enzyme .422

46.1.2 The Effectiveness Factor 422

46.1.3 Maximal Effective Enzyme Loading 423

46.1.4 Decline ofVmax0 Over Time 423

Part IX Special Statistics

47 Quality Control 427

47.1 Validation 428

47.2 Assessing the Quality of Measurements 431

47.3 Analytical Results Need Careful Interpretation .432

47.4 False Positives in Large-Scale Screening 433

48 Testing Whether or Not a Model Fits the Data 435

48.1 The Runs-Test 436

Part X Appendix A List of Symbols 441

B Greek Alphabets 445

C Properties of Electrophoretic Buffers 447

D Bond Properties .453

E Acronyms 455

References 465

Index 487

Part I

Analytical Techniques

Chapter 1

Microscopy

The microscope is without question the most important instrument available to thebiologist The physiological function of proteins cannot be addressed without tak-ing their localisation in a living cell and their interaction with other proteins into

micro.magnet.fsu.edu/primer/covers microscopic techniques in much more detailthan possible here

1.1 Optical Foundations of Microscopy

Objects in cell biology range from an ostrich egg (20–30 cm) down to subcellular

can be seen with the naked eye, magnification is required for others

1.1.1 K ¨OHLER-Illumination

The microscope is a system of lenses which create images of the illumination and

is created; in the object planes an image of the object As you can see from thefigure, light from each point of the light source is spread evenly across the entireobject planes, passing the object in parallel beams from every azimuth This cre-ates a homogeneous, bright illumination; thus only a low power light source (lowvoltage halogen lamp of about 20 W) is required The apertures have to be adjusted

in position and diameter to avoid contrast reduction by stray light and to achievemaximum resolution

1.1.1.1 Critical Illumination

or clinical laboratories In this case the image of the light source (the frosted glass

E Buxbaum, Biophysical Chemistry of Proteins: An Introduction

to Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 1,

© Springer Science+Business Media, LLC 2011

3

Fig 1.1 Size of objects in cell biology, compared to the wavelengths of different electromagnetic waves

of a light bulb) is projected into the object plane by the condenser Such scopes are easier to use since condenser height and field diaphragm need not bereadjusted each time the objective is changed; they are also considerably cheaper.With modern, multilayer-coated lens systems the increase in stray light and reduc-

system is not suitable, since “hard” (high contrast) films amplify any inhomogeneity

in the illumination

1.1 Optical Foundations of Microscopy 5

Fig 1.2 Schematic diagram

of a microscope with the

illumination system originally

introduced by A UGUST

K ¨ OHLER at Zeiss The lens

systems create images of both

the object and the light

source The illumination

apertures adapt the numerical

aperture of the illumination

system to that of the

objective The apertures in

the object plane adjust the

field of view Thus the

diameter of the light beam

and its opening angle can be

adjusted independently This

reduces the stray light inside

the microscope Careful

adjustment of the size and

position of the apertures is

required to take full benefit of

the microscope

Collector

Light source

Condenser Object Objective

Ocular intermediate image

Pupilla Lens Retina

Condenser aperture

} Eye

Objective aperture (virtual)

Field diaphragm (in eye piece diaphragm)

Mirror

1.1.2 The Role of Diffraction

Two factors influence the power of a microscope: resolution and contrast Contrast

can nearly always be increased by staining or by optical methods (see later), the

microscope only needs to keep the level of stray light down The resolution (the

minimal distance between two objects that still allows them to be seen as separate),however, is subject to tight physical limitations

Responsible for the image formation is the process of scattering the light waves

on the object The scattered light creates a primary interference pattern for eachpoint of the condenser aperture in the objective aperture of the microscope (see

diffraction maxima depends on the grid constant (distance between structures) of

the object

Edge parallel light beam

− 2 − 1 0 +1 +2

Fig 1.3 (a) If a light beam is passed through a pin hole, light beams at the edge of the hole depart from their path Simple beam optics can not explain such behaviour (b) The same situation

viewed by wave optics A linear wave front (equivalent to parallel light beams) reaches a wall with

a pin hole Scattering results in a curved wave front (c) If there are two pin holes, the resulting

wave fronts interfere with each other Out-of-phase waves cancel, in-phase waves amplify each other Thus a pattern of bright and dark rings becomes visible The central bright disk is called

the maximum of zeroth order, the surrounding rings are numbered first, second order (d) The

light intensity plotted as function of the position in the interference pattern The maximum of zeroth order is much brighter than the first, higher order maxima are even weaker Note that the interference rings are symmetrical, thus each ring results in two peaks, one to the right (positive numbers) and one to the left (negative numbers) of the centre

Since the light from these diffraction maxima continues to travel upwards, and

since it comes from a single point, the beams are capable of interference This

creates a secondary interference pattern, the intermediate image, which is then

www.doitpoms.ac.uk/tlplib/diffraction/index.php

1.1 Optical Foundations of Microscopy 7

The simplest object is a tiny hole Its image is a pattern of bright and dark rings,called AIRY-pattern

1.1.3 The Importance of the Numerical Aperture Na

to the object, the more diffraction maxima are collected for it The diffraction imum of zeroth order contains most of the light, but no information, since it has notinteracted with the object This means that to reconstruct the object in the interme-diate image, at least the diffraction maximum of first order must be in the objectiveaperture Since finer object details give a higher distance of the refraction maxima

max-from each other, the resolution capability of a microscope depends on the objective

aperture

The radius of this aperture is calculated to

with˛ being the opening angle of the light cone and nm the refractive index of

the medium Optically important media and their refractive indexes can be found in

Table 1.1 Refractive index n

Water 1.330 Glycerol 1.460 Fused silica 1.462 Toluene 1.489 Glass 1.520 Immersion oil 1.520

0 0.2



2

2mCa

(1.4)

The -function is a generalisation of the faculty-function for real arguments

the AIRY-pattern is

r D 0:61

d The radius of the AIRY-disk of a point is

We can imagine that each point of an object results in its own AIRY-pattern, sulting in a mosaic image Experience shows that two points are seen as separate if

between the overlapping disks has about 80 % of the intensity of the maxima ever, due to optical imperfections the radius of the AIRY-disk is somewhat largerthan that calculated above, empirically a factor of 112 % is assumed

1.1 Optical Foundations of Microscopy 9

Lens

d2

Fig 1.5 Lenses have a limited depth of focus While the image of the tip of the arrow is focused

on the screen, the image of the bottom is not Instead, it is drawn out into a dispersion spot with diameter d 2 This effect is stronger in stronger lenses with shorter working distance

n2 N2 a

(1.8)

2Na2

(1.9)

of “empty magnification” Magnification compares the angular size of images at adistance of 25 cm and is hence a rather arbitrary parameter

Remember It is the numerical aperture, not the magnification of an objective that

is really important

The numerical aperture of the condenser should be equal to that of the objective,thus condensers have a diaphragm This allows adjustment of the condenser aper-ture to the aperture of the objective Although this setting would give the highestresolution, in practice the condenser aperture is set slightly lower than that of theobjective This increases the depth of focus Since most objects are more or lessspherical this means that their borders in the image become thicker and more visible

1.1.4 Homogeneous Immersion

Since the maximum angle of the opening cone of an objective is 180° and the

1:0, in practice 0:95 For this reason some systems are corrected for the use of

im-mersion oil between object and objective (in some cases also between condenser

Na  1:4 (see Fig.1.7) Such systems are marked “Oil” and should not be used

Image with condenser open Image with condenser closed

Focal planeCell

Fig 1.6 Closing the condenser aperture a little more than required for K ¨ OHLER -illumination

in-creases the depth of focus of the image (dotted lines above and below the focal plane), resulting

in thicker, darker outlines of structures For an optimum balance between apparent contrast and resolution, condenser aperture should be 20–30 % smaller than that of the objective

without immersion oil as the resulting image would be very poor For observationunder UV-light, fused silica is used for the lens and the coverglass, in this caseglycerol is used for homogeneous immersion

1.1.5 Lens Aberrations

Chromatic aberration is caused by dispersion, that is, different refractive index

wavelengths, and in addition these images have different magnification A lens madefrom normal glass refracts blue light stronger than red The error is corrected byusing lens systems rather than single lenses, and the various lenses are made from

different materials Achromatic lenses are corrected for red and blue light Since

green light can easily be removed by a strong red filter, such relatively cheap lenses

are often used in black and white photography Apochromatic lenses are corrected

for four different wavelengths: deep blue, blue, green, and red Such systems areused for colour photography However, they are not only very expensive, the high

1.1 Optical Foundations of Microscopy 11

lost light

coverglass

coverglass

Fig 1.7 Top: Relationship between working distance, numerical aperture, brightness, and

resolu-tion of an objective, the object is a single bright point Systems with high numerical aperture collect more light and give smaller A IRY-disks (= higher resolution) Bottom: If the air between the cov-

erglass and lens is replaced by immersion oil with the same refractive index as glass, refraction of the light beams is avoided and a higher N abecomes possible (homogeneous immersion)

number of lenses inside also causes increased stray light, thus reducing contrast.Fluorite systems are in between achromats and apochromats The use of new glassmaterials in the last 30 years has improved the quality of achromats so much thatthose made by premium manufacturers are sufficient for all but the most demandingapplications

Spherical aberration is caused by lenses having different focal lengths for light

beams entering at the centre and in the periphery, even for monochromatic light.Again, this is disturbing mostly in micro-photography, during observation we can

simply play with the focus Lenses corrected for spherical aberration are called plan.

Modern lens systems from reputable manufacturers are so good that an investmentinto plan lenses is not usually required

Fig 1.8 Correction of lens

1.1.6 Special Methods in Light Microscopy

An object can be thought of as a simple grid that creates an interference pattern in

zeroth order is brightest, but its light has not interacted with the object and hencecontains no information The final image produced by the microscope depends on

in-cluding zeroth order, are allowed to form the image Resolution of bright-fieldmicroscopy is limited by the RAYLEIGH-condition: objects are visible in a mi-croscope only if they have at least twice the size of the wavelength of the light

stop in the condenser, which is projected into the objective aperture Objectsappear bright on a dark background Note that in dark field (and fluorescence)

1.1 Optical Foundations of Microscopy 13

Fig 1.9 The role of

interference in the formation

of the microscopic image If

no object is present each point

in the condenser aperture is

projected to one point in the

objective aperture If an

object (say a grid) is present,

it creates a diffraction pattern

for each point of the

condenser aperture in the

objective aperture, and their

interference creates the

Fig 1.10 Condenser filter for special effects Top: A darkfield filter stops light of zeroth order,

whilst a R HEINBERG -filter colours zeroth order light differently from that of higher orders In

darkfield polarisation light of zeroth order is polarised Bottom: Filters for oblique and oblique

darkfield illumination

microscopy the RAYLEIGH-condition does not apply, hence the term

ultrami-croscopy is sometimes used.

form the picture In theory this doubles resolution, but contrast improvement byshadowing is more important In science this method is rarely used since we cannot distinguish between shadowing by different refractive index and differentthickness

re-moved

Rheinberg contrast Different colour filters for light from the zeroth order andhigher order maxima Of aesthetic value only.

a different refractive index from the medium This results in a phase differencebetween the undiffracted (zeroth order maximum) and the diffracted light (higherorder maxima) If a phase plate is used to delay the zeroth order maximum (andreduce its intensity), subtractive interference occurs when the intermediate image

is formed This method is of extreme importance for the observation of living (unstained) cells.

linear polarised light A second polariser filter is mounted on the eye piece Thedirection of this filter is placed at right angle to that in the condenser, so that alllight from the light source is absorbed by either one filter or the other, creating adark background Any birefringent object will turn the direction of the polarisedlight and appear bright Biological examples for birefringent objects are muscleand starch granules In the lab protein crystals are birefringent Geologists andmaterial scientists use polarisation microscopes routinely

Polarisation and darkfield microscopy may be combined if only zeroth order light

is polarised This is useful for the observation of zooplankton

the emitted light has a longer wavelength, it can be separated from the ing light by a filter Objects appear bright on a dark background, with very

method, small amounts of fluorescent compounds can be detected (vital stains,immunofluorescence microscopy) Illumination is usually through the objective

layers of coating of different refractive index At the interface between these ers part of the light is reflected, the rest is refracted Depending on the thickness

lay-of the layers the reflected and refracted light lay-of some wavelengths undergo structive, of other wavelengths destructive, interference Therefore, the mirrorreflects some wavelengths, but transmits others

con-The high light intensity required for the exciting beam results in phototoxicity,which can be reduced by stroboscopic illumination, or by adding protective sub-

stances into the mounting medium (e.g., ascorbic acid).

2 photons and hence excite fluorophores with half the exciting wavelength

In-frared lasers (titanium sapphire) are used for excitation Because absorbtion oftwo photons by the same dye molecule within the lifetime of the excited state is

condenser Thus the emission comes from one point of the sample only, similar

the sample to get a 3-D image

1.1 Optical Foundations of Microscopy 15

A- Light source (halogen lamp)

A condenser aperture

A’ objective aperture A" eye pupilla

O- illumination

O Object

O’ Intermediate image O" Eye retina

Condenser

Objective Field lens Occular Eye lens

the dichroic mirror (which is transparent to long wavelengths), into the ocular and the eye Light

from the halogen lamp in the foot of the microscope (yellow beam) can be used to compare the

fluorescent with the transmission image

measured and used to sort cells (or other objects) into groups The number ofobjects in each group is counted; additionally it is possible to purify cells with

particu-lar at high magnification Light from out of focus planes creates a haze in the

from a focused object passes through a certain point along the optical axis In

In addition, he used a small light point to excite fluorescence and moved the

sam-ple under that point in x- and y-direction Thus weakly fluorescing objects could

be seen next to very bright ones The idea was ahead of its time: it simply tooktoo long to scan the sample The invention of the laser changed this; scanningthe laser across the sample can be done relatively quickly; lasers are also verybright and can be focused onto a small spot The fluorescent light is detected by

Fig 1.12 The confocal microscope Top left: Light coming from an in-focus object passes though

a single point Only a small fraction of the light from objects higher and lower than the in-focus object also pass through that point A pin-hole at the focus thus removes most of the light coming from out-of-focus objects The diameter of the pin hole should be the diameter of the A IRY -

disk Top right: Schematic diagram of a laser scanning confocal microscope The light from a

monochromatic source (usually a laser) is brought into the optical path via a dichroic mirror Two oscillating mirrors move the beam across the sample, and align the resulting fluorescent light with the optical system After passing the dichroic mirror the light goes through the pin-hole (which removes out-of-focus light) to a photomultiplier tube The resulting electrical signal feeds through

an analog/digital converter (ADC) into a computer system Scanning of the laser beam prevents fluorescent light from nearby objects in the focus-plane from entering the optics, increasing lateral

resolution Bottom NIPKOW confocal microscope: The laser light is spread to illuminate a section

of a rotating N IPKOW -disk Light passing the holes is focused onto the sample by the objective Fluorescent light emitted by the sample is focused by the same objective lens onto pinholes in a second N IPKOW -disk Thus several sample points are imaged onto a CCD-array at the same time, increasing the frame rate

object is moved up or down a little and the scan is repeated After several suchscans a three-dimensional representation of the object is created in the computer,which contains quantitative information on the light intensity in each point of theobject

1.2 The Electron Microscope 17

To further increase the rate at which pictures can be taken, several points of theobject may be scanned at the same time using a rotating NIPKOW-disk Suchsystems can either use lasers or white light for illumination

light onto the specimen shows diffraction, spot size is limited to about 200 nm,this limits the resolution of the confocal microscope In newer instrumentstwo laser beams are coupled into the microscope, one to excite fluorescenceand one to stimulate the emission of fluorescent light (note: do not mix upstimulated emission, which is reversible, with irreversible bleaching) Theemission-stimulating beam has zero intensity in the centre but high intensity

smaller than the diffraction spot The new resolution becomes:

increas-ing computer performance have now made it possible to remove the haze ofnormal microphotographs This is called de-convolution of the image Light fromdifferent depths is transferred through a lens system in different ways This can

be described mathematically using point spread functions, applying them in

reverse de-convolutes the image

fluorescence to a single plane The light is focused not into a point, but a sheet

by a cylindrical lens The light sheet enters the sample from the side Since onlyone plane is illuminated, out of plane light can not lower contrast Contrary toconfocal microscopy an entire section is captured at once, and scanning is re-

quired only in z-direction This high scanning velocity allows real time videos

objects to be studied (e.g., entire fruit fly or fish embryos in developmental

bi-ology) Also, phototoxicity and fluorescence bleaching are reduced because theexciting light is distributed over a much larger area

1.2 The Electron Microscope

1.2.1 Transmission Electron Microscopy

Visible light has wavelengths between about 380 (violet) and 780 nm (dark red) Ascan be seen from (1.6), the resolution of a microscope is inversely proportional tothe wavelength of the light used In normal bright field microscopy, the separation

distance is about twice the wavelength (i.e., 0:8 m min.), in dark field and

fluores-cence microscopy slightly lower separation distances are possible

Because electrons can behave like electromagnetic waves with very short length, it is possible to construct microscopes that use an electron beam rather than

emission electron microscopy), focused by a WEHNELT-cylinder and accelerated

bio-logical samples, 80–120 keV are most often used Magnetic fields are used instead

of glass lenses These fields are created by coils, the refracting power is determined

by the current flowing through these coils

Electrons can travel only in high vacuum, thus all objects observed in an EMmust be completely dry This limits EM to fixed, dehydrated samples Some attemptshave been made to circumvent this limitation (“wet EM”) In that case the objectsare mounted onto a thin, electron-transparent plastic foil and illuminated by theelectrons through the foil Back-scattered electrons also pass through the foil beforebeing collected to form the image

the sample during observation and cause artifacts Special stabilisation procedures(which are expensive and time consuming) can be used to limit this problem In spe-

with liquid nitrogen

Because electrons do not travel far in solid materials, samples need to be verythin Biological materials are usually embedded in plastic, cut on an ultramicrotomewith knifes made from glass or diamond, and placed on small copper grids Contrast

is increased by staining with salts of heavy metals like lead, osmium, or uranium

1.2.1.1 Cryo-Electron Tomography

glass-like) rather than crystallises, cell structures down to protein complexes are served The object is then scanned in the electron microscope under low-dosecondition and under constant cooling It is rotated, so that pictures are taken fromseveral angles From these tilt-series, three-dimensional reconstructions of the ob-ject are made in a computer using similar techniques as in X-ray tomography inmedicine The structures visible then need to be identified–that is, they are automat-ically compared with known structures (template matching)—and known proteinsare docked into the complex Identified components can be marked, for example, bycolour coding With this technique a resolution of about 5 nm is achievable Noisereduction in the cameras used will probably push that limit in the coming years For

1.2 The Electron Microscope 19

Fig 1.13 Raster scanning electron microscopy (first three pictures) and transmission electron

microscopy (last three pictures) of the liver wort Marchantia polymorpha The pictures show a leaf

with vegetative buds and a single cell, with nucleus and chloroplasts, the latter containing starch granules

1.2.2 Scanning Electron Microscopy

three-dimensional information is obtained from the sample This is an advantage

and the screen of the monitor tube The intensity of the beam in the monitor (andtherefore the brightness of a particular spot) is controlled by the number of electronsscattered from the sample into a detector in the microscope

Thus the brightness of a spot on the monitor depends on the relative positions ofthe electron gun, sample, and detector, and on the surface of the sample

The energy of the reflected electrons depends on the chemical composition of thesample; it is therefore possible to analyse the sample and quantitatively determinethe chemical elements that occur in it However, this method is neither sensitive norparticularly precise

Again, a high vacuum is required inside the microscope, and samples must fore be dried Drying must be done so as not to change the surface of the sample.This is achieved by replacing the water in a biological sample first with organic sol-

above its critical point (to about 45 °C) Above the critical point the phase border tween liquid and gas vanishes The gas pressure is then slowly reduced (so that thetemperature does not drop below the critical point!) through a valve The sample islater coated with a very thin layer of gold to make it conducting

be-1.2.3 Freeze Fracture

1.3 Other Types of Microscopes

To study specimens of molecular dimensions special techniques were developed

in the last 30 years, leading to a revolution in biology and (even more) material

electron microscope) “Microscope” is actually a misnomer for these instruments,since they do not look at the sample, but “feel” its surface

1.3 Other Types of Microscopes 21

(1)

(2)

(3)

(4) (5)

Fig 1.14 Freeze-fracture (1) The sample is vitrified in water (2) The block is fractured so that

the break occurs between the leaflets of a membrane The resulting block is then etched under a

vacuum, so that some of the water on the surface sublimates away (3) The surface is sputtered

with platinum from an angle (usually 45°), so that elevations on the sample cause shadowing.

(4) The platinum layer is reinforced with carbon, which is translucent to electrons (5) The block

is immersed in water, the original sample washed away, and the platinum/carbon film fished from the surface with a sample net for TEM

1.3.1 The Atomic Force Microscope

generated by the specimen on the tip is measured and minimised by a feedback cuit, keeping the tip just above the specimen Thus the elevation profile (z-direction)

cir-of the specimen is recorded By increasing the force generated by the tip it is sible to move atoms or molecules around (nano-technology) About 1–2 min arerequired for a complete scan, resolution is about 1 nm

surface properties like elasticity, charge or the presence of binding sites for ligands

(lab on a tip) By measuring the force required to dissociate the interaction between

Protein unfolding can be followed by binding the protein to both the support andthe tip and then slowly increasing the distance between tip and support The forcerequired to break bonds is recorded

1.3.2 The Scanning Tunnelling Microscope

(sub-atomic) distance above the specimen Between the tip and specimen a age is applied, and because of the small distance between them electrons can tunnel

volt-from one to the other The current thus generated is measured and kept constant bychanging the distance between probe and specimen using a feedback mechanism.

current through the protein can take several paths of different conductivity Thus thesignal obtained reflects not only the height of the protein at a particular position.With both techniques it is possible to investigate unfixed specimens underphysiological conditions Despite their atomic resolution the construction of suchmicroscopes is quite simple and can be done by any reasonably competent me-

chanic Both construction plans and software are available on the internet (e.g.,

http://www.geocities.com/spm stm/Project.html), putting these instruments into thereach of ambitioned high schools or amateurs

1.3.3 The Scanning Near-Field Optical Microscope

is a form of fluorescence microscope where the excitation beam is sent through aglass fibre The fibre is tapered at the end to a diameter of only a few nanometers.This end is scanned over the sample, illuminating only a tiny spot The fluorescentlight emitted by the sample is collected by a photomultiplier The resolution of theinstrument is therefore limited only by the diameter of the probe tip

In such a thin fibre the light no longer undergoes total reflection, therefore theoutside of the fibre must be coated with aluminium Once the fibre diameter becomessmaller than the wavelength of the light, light propagation is by tunnelling This part

of the probe therefore needs to be very short

Chapter 2

Single Molecule Techniques

Any experiment performed on this sample of protein, say, the determination of itsaffinity for a ligand, will return a value averaged over a huge number of molecules.That is fine as far as it goes, but what is the distribution? Is it normal or skewed?What is the standard deviation? To answer these questions, one has to perform ex-periments on single molecules and repeat those on many different specimens Onehas to determine what percentage of time a protein molecule has or has not ligandbound at a given concentration to determine the affinity Or one has to measure theforce required to pull a ligand from a protein molecule to determine the bindingenergy Such measurements are of particular interest for proteins that move What

is the torque generated by rotation in ATP-synthase? Can it explain the synthesis ofATP? How much force does a single myosin head generate when moving along anactin fibre? Only in the last 20 years have we started to answer such questions

2.1 Laser Tweezers and Optical Trapping

It is immediately apparent that–in a homogeneous sample in dynamic equilibrium–if

a certain fraction of molecules has ligand bound, then each molecule has ligand

molecule with its ligand becomes:

be vectorially added for all photons acting at a given moment

E Buxbaum, Biophysical Chemistry of Proteins: An Introduction

to Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 2,

© Springer Science+Business Media, LLC 2011

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