Methods in Modern Biophysics

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Add to Basket. Book Description Springer, Condition: New. Never used!. Seller Inventory PX. More information about this seller Contact this seller. Items related to Methods in Modern Biophysics. Methods in Modern Biophysics. Publisher: Springer , This specific ISBN edition is currently not available. View all copies of this ISBN edition:. Synopsis About this title Incorporating recent dramatic advances, this textbook presents a fresh and timely introduction to modern biophysical methods. Special features in the 2nd edition: - Illustrates the high-resolution methods for ultrashort-living protein structures and new results for 6 proteins.

From the Back Cover : Incorporating dramatic recent advances, this textbook presents a fresh and timely introduction to modern biophysical methods.

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Review : From J. It is best suited as a textbook for both undergraduate and graduate students for its richness in graphics and comprehensiveness of the text, but will certainly be adored also by researchers and lecturers. The book guides with ease the reader through its eleven chapters giving a near complete picture of current biophysical techniques Kaftan, F. Buy New Learn more about this copy. Other Popular Editions of the Same Title. The tags should not cause artificial interactions and should not alter the conformation of the tagged protein.

Very common are poly-histidine tags that are attached to the protein by genetic engineering Fig. The tag typically consists of 8— 12 histidine residues. It binds to nickel compounds at the surface of the chromatography beads. Antibodies with high binding constants bind to the antigen and are eluted later with a buffer with a high salt concentration Fig. About 10 histidine residues were attached to the protein by genetic engineering, e. The histidine residues strongly bind to the bead made from a nickel chelate resin 2.

The chaperone interacts with the sample protein and catalyses its folding into the correct conformation 2. Nowadays it serves for the large-scale purification of some chemicals for which other chromatographic methods are too expensive. As in other types of counter-current chromatography, both stationary and mobile phase are liquids and separation is based on sample partition between the two liquids. It may, e. Sample separation is based on its partition between the two liquid phases see text Fig. Pressurized nitrogen from a nitrogen flask presses the protein solution against the membrane.

Small molecules pass the membrane and are collectable at the outlet. Large molecules stay in the ultrafiltration vessel of impurities is transferred to the next apparatus and fresh phase B is transferred from the previous apparatus to the shown apparatus. During suc- 2. Pressure is applied by centrifuging the concentrator. Similarly to the pervious ultrafiltration device Fig. Strictly speaking, ultrafiltration Figs. However, it should be mentioned here since it is an extremely useful tool of sample preparation prior to chromatography and can sometimes even substitute chromatography.

It is applicable for a protein purification, b buffer exchange, and c concentrating protein solutions. Purification of a protein with a particular molecular weight, Mw , requires two steps: a First, one runs the ultrafiltration apparatus with a membrane with a cut-off higher than Mw and collects the solution leaving the vessel.

It is particularly useful for the detection and analysis of traces of macromolecules down to less than 1 pg 10—12 g. The general design of a mass spectrometer comprises sample injector, sample ionizer, mass analyzer and ion detector Fig. First the sample is injected into the ionizer which ionizes sample molecules. Then sample ions are analyzed and detected. To prevent collisions with gas molecules, sample ionizer, mass analyzer and ion detector are generally operated in vacuum.

R typically ranges between and , Time-of-flight mass spectrometers TOFs often are less expensive than other types of mass spectrometers and have, compared to quadrupole MS and many sector MS, the advantage of recording the masses of all ions injected into the analyzer without scanning, contributing to a high sensitivity. Ions leaving the ion source are accelerated and passed through the sector in which the electric or magnetic field is applied perpendicular to the direction of the ion movement. In scanning mass analyzers a the electric or magnetic field strength is varied and only one mass detected at a time.

In non-scanning mass analyzers b all masses are recorded simultaneously within a limited mass range with the help of a multichannel detector Fig. The ion beam is accelerated to a high velocity by an electric field and passed through the quadrupole mass analyzer comprising four metal rods. After passing through the accelerating potential difference, V, the kinetic energy, E, of an ion with the charge, z, mass, m, and velocity, v, is: 3.

A mass resolution of 1 Da requires in this example a time resolution of 3. Unfortunately, not all ions start to move at the same time and not all ions have the same velocity. The differ- Fig. The linear configuration of TOFs represents the simplest implementation of the time-of-flight technique.

Due to the chromatic aberration and the differences in the starting time, the requirements for a very high resolution are hard to meet in the simple design of a linear TOF Fig. In reflectron TOFs Figs. The reflector enhances mass-spectrometric resolution: it increases the time of flight and can focus ions. Here a voltage pulse at the shutter electrode causes a uniform starting time of the ions Fig. For further details see, e. The magnetic field forces the thermal ions on orbits with small radii that depend on their mass-to-charge ratio.

For the method of Fourier transform see also Sect. The striking characteristic of FTMS is the high resolution, R, typically in excess of , 44 3 Mass spectrometry 3. Ions are mainly guided by electrostatic lenses and quadrupole or octopole ion guides Fig. With the exception of FTMS, the ion signals emerging from the mass analyzer of the MS are commonly detected with an electron multiplier Fig. In FTMS the cyclotroning ions are indirectly detected by measuring and Fouriertransforming the voltage signal they induce into receiver electrodes.

Analyte solutions delivered by liquid chromatography or a syringe pump are sprayed through the narrow, heated capillary leading into the mass spectrometer. A voltage of typically V — 5 kV is applied between capillary and orifice in front of the electrostatic lenses. Ions form in vacuum by evaporation of the analyte solution of charged droplets Fig. Compared with a quadrupole ion guide, it enables a higher precision of guidance 3.

The first dynode converts the ion current into an electron current. Further dynodes amplify the electrons by a total factor of typically —, largely dependent on the electron accelerating voltage between the dynodes, the number of dynodes, and the dynode composition. The last dynode is connected with an ammeter not shown 3. The two types of connectors between chromatography and MS shown in Figs. Two-dimensional spectra are obtained through Fig.

The resolution in two dimensions greatly enhances the analyzability of complex mixtures with a large number of components. For example, ion exchange chromatography on a crude cell extract with a resolution of combined with mass spectrometry with a resolution of 10, can result in a total resolution of almost 1,, for small and medium-sized soluble cellular proteins for which both methods are often largely independent from each other.

Buffer interference which is occasionally observed in MS can usually be prevented by increasing the sample concentration, decreasing the buffer concentration, or changing the buffer Fig. The combination of mass spectrometry with gas chromatography can greatly enhance the resolution of complex samples Fig. In several stages the solvent is removed from the analyte solution by application of dry nitrogen and vacuum. The quadrupole ion guide leads the ions to the mass analyzer of the mass spectrometer Fig.

Left: a number of side-peaks indicate the binding of buffer ions to the highly charged protein. Right: a measurement with a lower buffer concentration and higher protein concentration at a pH closer to the pI of the protein yields a cleaner mass spectrogram 3. These particles are then decomposed by pyrolysis see Fig. The device splits the total flow of the aerosol into the minor and the major flow. Because the direction of the major air flow is perpendicular to the original direction of air flow, only particles with sizes smaller than the cut-off size can enter the major flow.

In contrast, all large particles with a large inertia remain in almost the original direction of flow and join the minor flow containing also some of the small particles Marple and Chein, ; Marple et al. The sensitivity of some of these detectors is better than 1 biological agent particle per liter of air at a detection time of less than 3 minutes. The current rapidly heats the ferromagnetic foil up to the Curie point where the foil reversibly ceases to exhibit ferromagnetic properties and further heating stops.

Suitable ferromagnetic materials are, e. Pyrolysates generated in vacuum are then transferred to the mass spectrometer. The pyrolysis mass spectrometry PyMS method Aries et al. In this method the sample is partially decomposed in its components prior to massspectrometric analysis.

The mass spectra of pyrolyzed biological systems may contain more than lines, enabling a very sensitive differentiation of different samples. PyMS is used for the detection of bacteria, bacterial spores and viruses and the differentiation between different species of bacteria and viruses Freeman et al. PyMS spectra may be analyzed by using neuronal networks Fig.

Ion trap mass spectrometers are particularly suitable for the pyrolysis-MS identification of biological agents since they can directly measure multiple fragmentation Fig. The fragmentation is achieved, e. In this simplified example the network consists of only 10 input, 5 hidden, 3 output nodes, a bias, and weighted connections. The hidden layer may actually contain 8 — 20 nodes. Preferentially the weights of the connections and the bias are set by supervised learning using the hazardous substances to be detected or simulants of these hazardous substances Fig.

Top: amplification of the template DNA with termination only one of the four reactions is shown. Middle: for comparison to the mass-spectrometric method: conventional analysis of the reaction products with polyacrylamide gel electrophoresis. Bottom: analysis of the products of reaction A by mass spectrometry: the pattern of mass peaks shows the positions of adenine A in the sequence 56 3 Mass spectrometry Fig. This protein preparation contains a fraction with a molecular weight Da higher than expected. This is due to an N-terminal methionine which is not properly cleaved after protein synthesis Fig.

In this example with only three proteases, only some parts of the peptide sequence can unambiguously be identified 3. This sketch illustrates a mobile with mini-TOF searching for extraterrestrial life. Already now mass-spectrometry is utilized to search for extraterrestrial bacteria in meteors 4 X-ray structural analysis 4. Since the technique of Fourier transform is central to this method, we first deal with some essential aspects of this technique: 4. This decomposition was calculated from the Fourier transform c. The inverse transform of c yields back exactly a.

The comparison of d with a illustrates the importance of the phases in Fourier transform: in order to be able to correctly obtain back the original signal by inverse Fourier transform, both the amplitudes and the phases have to be known. Three examples in Figs. In Fig. Fourier transforming a noisy object, then substituting certain low-amplitude parts of the Fourier transform by zeros, and then inverse-transforming the modified Fourier transform, is an efficient method for noise reduction.

However, when adding the four components with the wrong phase 0, we obtain the wrong sum d. Only the absolute of the function is drawn in b. However we must keep in mind that the complete function contains an amplitude and phase for each coordinate point compare with Fig. Note that in this figure only the absolutes of the functions are drawn 4. Note that in this figure only the absolutes of the functions are drawn 64 4 X-ray structural analysis Fig.

The X-ray diffraction pattern of the crystal is recorded with an area detector. The pattern consists of a large number of discrete spots Why is the Fourier transform so important for X-ray crystallography? This is because the diffraction pattern of a crystal Fig. To understand why the diffraction pattern of a physical object is its Fourier transform let us consider the diffraction of a wave by a single object Fig.

Then the phase difference in units of wavelengths between the two waves in Fig. Hence the electron density and structure of a protein can be obtained from the inverse Fourier transform of its diffraction image. Unfortunately, currently there is no X-ray microscope with sufficient resolution and sensitivity. X-ray mirrors do not provide sufficient resolution, and because of radiation damage, we would not obtain a satisfactory resolution for a single protein molecule anyway. That is why we have to record the diffraction pattern of a protein crystal and to calculate the inverse Fourier transform of the diffraction pattern with a computer.

Unfortunately, when recording the diffraction pattern of an object with the help of a camera, all phase information is lost. With other 66 4 X-ray structural analysis words, we do not record the complete Fourier transform, but only a fraction of it. The consequences of this serious problem were illustrated in Fig. Thus, additionally to the recording of the diffraction pattern, one needs a special technique to recover the phase information.

The currently most important method to recover phase information in protein crystallography on new structures is the technique of heavy atom replacement see Sect. A specifics of the diffraction of macroscopic crystals is that not a continuous diffraction pattern is obtained, but discrete spots. To understand this behavior, consider the structure of a crystal Fig. It determines the maximum number and positions of the observed diffraction spots The protein crystal can be described as the convolution of the crystal lattice with the unit cell Fig. The unit cell is the smallest unit from which the crystal can be generated by translations alone.

The latter is called reciprocal lattice Fig. Since the reciprocal lattice is zero outside its lattice points, the crystal diffraction pattern corresponds to the Fourier transform of the unit cell sampled at the points of the reciprocal lattice. A second way to explain the occurrence of discrete spots in the diffraction pattern of macroscopic crystals, and to evaluate the information from the intensity of theses spots, is to think of the diffraction as a reflection on the X-ray at the lattice planes of the crystal Fig.

These lattice planes are described by the Miller indices Fig. Diffraction is viewed as reflection of the X-ray on the lattice planes Fig. The initial model is refined, e. It took three decades and the development of computers to obtain the first 3-D structures of proteins Kendrew et al. Many thousands of native protein structures have been solved since then. Examples are found in Figs.

A few structure determinations were even made under artificial conditions, e. Commercial crystal screening kits, containing the most prominent buffers for protein crystallization, may be obtained, e. Important parameters for coarse-screening and fine-adjustment are protein concentration, salt types and concentrations, pH, type and concentration of surfactants and other additions, temperature, and speed of crystallization.

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  • Crystals with cracks, intergrown crystals and crystals with cloudy inclusions are generally unsuitable for X-ray crystallography. Totally unsuitable are stacks of plate-like crystals or needle-like fibers and mosaics 70 4 X-ray structural analysis Fig. The solvent of a small drop of protein or virus solution attached to a cover slide slowly evaporates partially. At the right conditions, a single crystal of suitable size grows Fig.

    The computer-controlled mixer draws different solutions from reservoir bottles, mixes them with various ratios, and places the mixtures on a glass plate 4. Usually, the protein or virus must not contain a significant amount of contaminants, such as other proteins or viruses, protein or virus fragments, unfolded or misfolded protein, particulate matter, chemical additions unnecessary for stability or solubility.

    In most cases compact proteins that do not contain floppy ends, such as histidine tags or native unstructured peptides, crystallize better. Suitable crystals have sizes of a few 0. With the example of a linear CCD, Fig. The most common X-ray sources for protein and virus crystallographic analysis are rotating anode generators Fig. Synchrotrons are comparably expensive, but have a higher brightness enabling shorter measuring times.

    Reduction of the exposition time often results in a better quality of the diffraction pattern since decomposition of the crystal due to radiation damage is reduced. The highlighted section is referred to in Fig. The crystal is cooled with nitrogen from the cryogenic system nozzle. Cooling the crystal reduces radiation damage, but somewhat changes the intermolecular distances.

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    For reduction of the dark current, this CCD is operated at — 40 oC, allowing it to detect single photons. The fiber optic taper serves also for blockage of X-rays and thereby prevention of radiation damage to the sensitive CCD array. The sensor elements generate electrons by absorption of photons and store the electrons in potential wells. After a certain period of time, the collected electrons are transferred to the analogue shift register and read out. An electron beam is focussed onto the rotating anode.

    It knocks out electrons from the inner electron shells of the anode metal. Reoccupation of the vacant shells by electrons from higher level shells involves the emission of X-ray radiation. The interaction of the electron beam with the anode metal generates also a large amount of heat which is quickly dissipated by rotating the anode below the spot of incidence of electrons 4. Ions or electrons are accelerated to a speed close to the speed of light and forced on a curved trajectory. A broad spectrum of radiation is produced along the curved sections of the beam.

    For protein crystallography, a certain wavelength, e. The dashed circle indicates the area corresponding to a resolution of 2. If diffraction spots would be visible up to this circle, the resolution of the obtained structure would be 2. One can see that the resolution is much lower in this example Already superficial inspection of the diffraction pattern provides a lot of information about the quality of the crystals: since the information about fine details of the protein structure is found at large diffraction angles, the absence of spots far outside the center of the diffraction pattern shows that only a low resolution will be obtained Fig.

    If we do not have information from molecules with a similar structure, or anomalously scattering atoms in the molecule, the method of choice may be the heavy atom replacement: the diffraction pattern of the original native crystal is compared with crystals that contain a single or a few heavy atoms at fixed positions. Those crystals can be prepared, e. This shows that they belong to phases with a large magnitude. How can we make this conclusion? See Fig. Essentially no change of intensity occurs at phases around zero. So, by comparing the intensities of the spots between native crystal and the heavy atom derivative we can estimate the phases of the individual diffraction spots.

    With only one heavy atom derivative, an uncertainty of two possibilities remains for each spot, but this can easily be removed with a further, different heavy atom derivative of the protein crystal. Right: heavy atom derivative Another way of showing the importance and meaning of phases in crystallography is illustrated in Fig. Thus, without information from heavy atom replacement, or from diffraction patterns of proteins with similar structure or other information, we cannot deduce the protein structure from the diffraction pattern.

    Theoretically one could also try out all possible phases and see if it leads to a meaningful structure, but currently for macromolecules the computational effort would be much too high. It should be noted that the problem of loss of phase information occurs only in the common methods of recording the crystal diffraction, such as with a photographic film or a semiconductor detector.

    The use of lenses or mirrors to produce an image like in an microscope would prevent this loss of information see p. Further, it is also very difficult to build highly precise X-ray mirrors Figs. X-ray mirror microscopes using soft radiation currently reach only a few 10 nm resolution. More importantly, the radiation damage would prevent atomic resolution of a single protein molecule or virus. Thus, e. Analogously one can estimate the phases from the observation of various intensity changes of diffraction spots upon derivatization of the crystal with a heavy atom.

    This uncertainty is removed by using data from a second heavy atom derivative Fig. From the imaginary and real parts Fig. The difference of the absolutes of Fourier transforms between native array and heavy atom derivatized array is shown in Fig. Comparing this difference of the absolutes of Fourier transforms with the absolutes of the phases of the native array Fig.

    This connection allows the magnitude of the phase angles to be determined. As mentioned, the remaining ambiguity of sign is removed by including the data from a second isomorphous heavy atom derivative. When we compare Figs. It may be found on the internet, e. Time- Fig. The CO is flashed off the heme group of the heme protein. This initiates a conformational transition which is detected, e. In cryocrystallography, reactions are induced and measured at a low temperature. At the very low temperatures of flash photolysis and acquisition of the diffraction pattern in the experiment shown in Fig.

    This enables to determine the coordinates of structural intermediates that would normally be too short-lived to be resolved by X-ray crystallography. Significant effort is undertaken to enable the measurement at very small angles. For details on X-ray optics see also the previous section. A large number of vials is automatically sampled and production faults immediately are detected and responded to 86 4 X-ray structural analysis SAXS measurements revealed that a an unliganded aspartate transcarbamoylase adopts a T-quaternary structure Fetler et al.

    SAXS revealed information regarding the conformational diversity and size distribution of unfolded protein molecules Kamatari et al. SAXS is one of the very few methods which can directly monitor structural changes of small virus particles Sano et al. The X-ray beam from a rotating anode generator is passed through a crystal monochromator that selects a wavelength.

    The monochromatic X-ray beam is passed though the sample cell, and scattered X-rays at very small angels are passed through a second crystal monochromator and then detected with a scintillation detector. For many applications, this set-up may be simplified, e. In some cases even low resolution solution structures of proteins were obtained solely from SAXS data Chacon et al.

    SAXS can reveal the structure of bones Rinnerthaler et al. SAXS results on human dentin, which is a complex composite of collagen fibers and carbonate-rich apatite mineral phase, are consistent with nucleation and growth of an apatite phase within periodic gaps in the collagen fibers Kinney et al. X-ray backscattering provides much better contrast in this application. However, the quantum efficiency of X-ray scattering is low and thus relatively large expositions and sensitive cameras must be used 4. A problem of detection of organic material, such as illicit drugs and explosives, by X-ray absorption is their low absorption coefficient compared with metals and the possibility to camouflage the material, e.

    X-ray backscattering offers a good contrast for the detection of such powdery material Fig. The main disadvantage is the low backscattering coefficient compared with transmission coefficient of most organic samples. Thus, a significantly higher exposure compared with X-ray transmission is usually required.

    Depending on the measurement technique, scanning infrared IR spectrometers, Fourier transform infrared FTIR spectrometers, and single wavelength infrared apparatuses are distinguished see Sect. Infrared activity requires a change of dipole moment upon excitation Fig. Regarding the resolution of protein secondary structure, the information content of IR and FTIR spectroscopy is comparable with that of circular dichroism see, e. Note that infra-red activity requires a change of dipole moment 92 5 Protein infrared spectroscopy 5.

    The emission of the source, e.

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    The monochromatic beam is split into two beams — one having the sample in the path. A shutter passes through only one of the two beams at a time. Both beams are alternatingly detected by an IR detector, e. The optical density of the sample is calculated from the logarithm of the intensity quotient. Spectra are recorded by scanning the wavelength region of interest. The monochromator separates the radiation of the IR source into its different wavelengths and selects one wavelength at a time. A beam splitter separates the monochromatic beam into sample beam and reference beam.

    The absorption coefficient, according to the chemical and structural properties of the sample molecules, is calculated using the detected intensity quotient between both beams, the pathlength, and the sample concentration 5. The better usage of radiation improves the inherent signal-to-noise ratio, especially for strongly absorbing samples for which the measurement may be photon shot noise limited. Also the spectral resolution of FTIR spectrometers, which is limited by the path length of the moving mirror, is often better than that of scanning IR spectrometers. Both the moving mirror and the fixed mirror reflect the beam back to the beam splitter which reflects the half of both beams to the detector where they interfere according to their phase difference.

    The light intensity variation with optical path difference, called interferogram, is the Fourier transform of the incident light spectrum light intensity as a function of the wavenumber. Absorption spectra are obtained by measuring interferograms with a sample and with an empty sample cell in the beam and inverse Fourier transforming the interferograms into spectra Figs. The lamp, e. A Michelson interferometer, consisting of a beamsplitter, a fixed mirror and a moving mirror, splits the beam into two beams and generates an interference of them. The sample inserted in one of the beam paths changes the interference.

    Interferograms with and without sample are recorded and the absorption of the sample is calculated by inverse Fourier transform see Fig. From the interferogram, the intensity of the beams can be calculated by inverse Fourier transform: 5. The absorption is given by the logarithm of the intensity quotient of blank to sample. Top: both beams have the same phase; their interference yields the maximum of the interferogram, i. IR intensities at the detector are recorded both for the sample cell filled with solvent and for the sample cell filled with sample.

    Inverse Fourier transform of the two interferograms yields the IR intensities. The IR absorption spectrum is calculated using the logarithm of the intensity quotient 96 5 Protein infrared spectroscopy Fig. The transparent walls of the cell are made from silicon wafers supplied by a manufacturer of electronic chips A very suitable material for the manufacture of sample cells, sample holders, and windows is silicon Fig. Polished silicon wafers of 0. Only the fragility and the high refractive index of this material might be problematical in some experimental set-ups. Used infrared sources are often thermal sources operated at about oC.

    Liquid nitrogen cooled mercury cadmium telluride MCT detectors and deuterated triglycine sulfate DTGS pyroelectric detectors are frequently applied for infrared detection. Since the light travels extremely fast, the detector senses the return echo before the next pulse is sent. The time it takes for the laser pulse to travel down and back is a measure of the distance.

    This method has importance, for example, for early warning systems of smog in large cities and for threedimensional analysis of forest structure and terrain Fig. Remote sensing of changes in forest structure utilizes the information of time and intensity of multiple reflections from leaves and branches. Effects of environmental pollutants and pests are quickly detectable in vast areas and economic damage is largely reducible.

    The latter contains several gyroscopes and an accelerometer and can determine the position and angle of tilt with some accuracy during periods of failure of the GPS Fig. The IR light from a light emitting diode is split into reference and probe beams. Light of the probe beam reflected from the sample is interfered with light of the reference beam, and the interference is detected by the photodiode.

    The pathlength of the reference beam is modulated by stretching an optical fiber with a piezoelectric transducer. Light from the sample which has traveled the same distance as the reference beam interferes constructively. Its signal is extracted from the interference intensity by a lock-in amplifier Duncan et al.

    Polychromatic backscattered light from different depths interferes with polychromatic light of a reference beam. The interference of the beams is analyzed with a polychromator and a multichannel detector. From the spectral changes due to interference, information about the depth of the scattering layer is obtained Another important variant of IR spectroscopy on biological samples is optical coherence tomography OCT. OCT Figs. In the design of Fig. Light from a scattering layer in the sample with a certain depth has the same phase as the reference beam and thus interferes constructively, i.

    Light from slightly deeper or shallower scattering layers cause a lower interference intensity. By modulating the phase of the reference beam and detecting the interference intensity with a lock-in amplifier, the signals from layers with different depths are extracted from the interference intensity Duncan et al. Here the information on depth is gained by analyzing the spectrum of the backscattered light. The internally total-reflected light slightly leaves the waveguide and so can probe the sample molecules on the outside of the waveguide.

    The part of the light wave which leaves the waveguide at the total reflection points is called evanescent wave. Only very little sample is needed. Using a large number of reflections can lead to a more than fold amplification of the measured signal 5 Protein infrared spectroscopy 5. Here the coefficient of internal total reflection of an IR beam in a waveguide is changed by a sample deposited on the surface of the waveguide. An advantage of ATR on thin layered samples is the dramatic increase of the effective optical pathlength and sensitivity through multiple reflections compared with conventional transmission spectroscopy on such a sample.

    The focussed beam from an IR laser is passed through the sample and detected. Left: simple microscope with planar resolution, especially suitable for thin layer samples. Right: microscope with three-dimensional resolution: for acquisition of the image, the sample is moved in xyz-directions 5 Protein infrared spectroscopy Because of the significantly lower scattering of IR light relative to light of shorter wavelength, IR microscopes Fig. Computer aided image processing allows two- or three-dimensional resolution.

    Since the spectrum of the isotope-labeled part of the protein molecule is significantly shifted, it can be distinguished from the spectrum of the non-labeled part. A change of the protein IR spectrum upon binding of the substrate to the protein shows which part of the molecule the substrate binds to. In this example, the magnitude of a peak in the spectrum of the isotopelabeled part of the protein has changed upon binding of the substrate. This shows that the substrate binds to the labeled part of the enzyme 5.

    Since the volume of unfolded protein is less than that of folded protein, high pressure favors transition to the unfolded state Fig. In some cases, interactions were resolved at the level of individual amino acid residues Kandori et al. Isotope-edited FTIR is particularly useful for the structural characterization of specific macromolecular regions Fig. Time-resolved step-scan FTIR spectroscopy enables the monitoring of conformational changes of proteins in the microsecond time scale Bailey et al.

    IR and FTIR spectroscopy are two of the only few methods suitable to monitor conformational changes of proteins under high pressure Fig. FTIR spectroscopy on bacteriorhodopsin revealed a pre-melting conformational transition at 80 oC Heyes et al. FTIR is also suitable to investigate the structure and hydration shell of protein molecules in organic solvents Fig.

    Molecular changes of preclinical scrapie can be detected by IR spectroscopy Kneipp et al. FTIR spectroscopy can serve as an optical nose for predicting odor sensation van Kempen et al. IR spectroscopy is also a tool for discrimination between different strains or types of cells Gaigneaux et al. Percentages of structure content and structural changes, e.

    The peaks around cm—1 and cm—1 correspond to the amide I and amide II bands, respectively. Such decompositions can be calculated, e. The sample was only two monolayers of a protein. Since at very low sample absorbances it is quite difficult to avoid the sharp lines of water-vapor absorption, these measurements were taken in a nitrogen-filled chamber at two different, very low concentrations of water, and later the water spectrum was subtracted. With this procedure, average artifact and noise levels were reduced to less than 0.

    In some aspects, the Fig. A typical objective lens has a bore of 2 mm and a focal length of about 1— 2 mm operation of a transmission electron microscope TEM is comparable with that of a slide projector Figs. The electron beam shines through the specimen. Objective lenses and projector lenses magnify the transmitted beam and project it onto the fluorescent viewing screen. Impact of electrons excites the screen and produces a visible magnified image of the sample. This image is recorded with various detectors, such as a CCD camera.

    Methods in Modern Biophysics

    High-resolution TEMs permit spatial resolutions around 0. Because of the wave nature of the electrons, the resolution limit, d, is given by the diffraction theory of coherent imaging: , 6. For a voltage of, e. A considerable concern is the brightness and size of electron sources. Thus, a large source can be focussed on a small spot only by using a large aperture angle of the optical system. Considering the limited aperture angles of electron lenses, a source of small size and high brightness is required to obtain a sufficiently bright picture of the sample.

    Usually they are made from conductive chemically inert non-gassing materials suitable for high vacuum, such as platinum and platinumiridium alloys. Among these materials, platinum is the chemically most inert, but expensive. Electron lenses have some similar characteristics like optical lenses, such as focal length, spherical aberration, and chromatic aberration. One of Ernst Ruska's major achievements was the development of electron lenses Fig. Additional coils may serve for the correction of spherical aberration not shown 6 Electron microscopy One important difference of electron optics compared with photon optics is the mutual charge interaction of electrons in the beam.

    That is why electron optics is often designed for beam paths with few if any intermediate crossovers. The high energy electrons are less affected by stray magnetic and electric fields. Also the propagation of the electrons in the column is independent from the selected electron probe energy.

    The electrons are accelerated to a high energy, passed through condenser lenses and filters, and then decelerated prior to interacting with the sample. This technique largely protects the electron beam against stray magnetic fields in the column of the microscope 6. The dispersion of electron energy is measured with energy filters Fig. Similar dispersive elements serve for the reduction of chromatic aberration, i. Inelastically scattered electrons have changed both direction and energy and may generate a diffuse contrastreducing background image unless these electrons are eliminated by energy filtering see Fig.

    Elastically scattered electrons interfere with another and with unscattered electrons to produce a phase contrast image Fig. The voltage applied between the two hemispherical electrodes disperses the electrons with respect to their velocity. In order to record a full spectrum for a large range of electron energies, the applied voltage is swept 6 Electron microscopy Fig. Right: filter using magnetic and electric fields Phase differences due to differences in the optical pathlength and electron scattering contribute to the contrast Fig. Often it is quite difficult to generate sufficient sample contrast at very high resolutions.

    A common method to visualize very small biological structures, such as single protein molecules, is negative staining: the sample is embedded in a stain with strong electron-optical properties Fig. Important innovations towards better contrast were the introduction of a technique for enhanced resolution Haider et al.

    For biological samples a further important reason for the limitation of the resolution of TEMs is radiation damage, i. Since a certain number of electrons is necessary to obtain an image, this limit depends on the ratio of inelastically to elastically scattered electrons. Negatively staining Fig. It was suggested the theoretical possibility of a neutron microscope, for which the ratio of elastically to inelastically scattered particles may much better for isotope-exchanged proteins Henderson, Another theoreti- 6.


    The electron beam is weakened at different points to a different degree by scattering and interference: elastically scattered electrons, i. The TEM structure is consistent with the crystal structure. Electron microscopy contributed to the understanding of conformational changes connected with the opening of an ion channel through a membrane Saibil, b , and with connexin trafficking Gaietta et al. In groundbreaking experiments Terry G. Frey and coworkers succeeded in the 3D-visualization of cell organelles using electron tomography.

    In this method the three-dimensional structure is calculated from a series of electron micrographs of samples tilted over a range of angles Dierksen et al. Bottom: the X-ray crystal structure of GroEL at 2. The resolution is generally limited by the diameter of the electron beam at the location of the sample and radiation damage. Scattered electrons are detected with the elastic dark field detector.

    The STEM image is generated by moving the focussed beam over the specimen Although the STEM was pioneered already in the thirties of last century, mainly by adding scan coils to a TEM von Ardenne, , significant developments have taken place in the last years: electron optics has been significantly improved and the resolution increased by several orders of magnitude.

    Nowadays high resolution STEMs offer unprecedented capability for the characterization of biomolecules, allowing structure to be determined with up to sub-nm resolution. A third mode is bright field detection where electrons are collected through a small aperture placed on the optical axis and an energy 6 Electron microscopy Fig. Electrons with different energies are separable by their trajectories with different curvatures in the electric field applied perpendicular to the flight direction filter removes those electrons that have lost energy, i.

    Quate in the mid-eighties Binnig et al. A very sharp tip attached to a tiny cantilever probes the sample surface. An optical system comprised of diode laser and detector, e. For scanning the surface, the sample is moved by the piezoelectric scanner Binnig et al. The AFM is used in both industrial and fundamental research to obtain atomic-scale images of metal surfaces and nanometer-scale images of the three-dimensional profile of the surface of biological specimens. It is a very useful tool for determining the size and conformation of single molecules and aggregates adsorbed on solid surfaces.

    The AFM scans the sample with a tiny tip mounted on a small cantilever Fig. It measures the small force of interaction between tip and sample surface by sensing the reflection changes of a laser upon cantilever movement caused by interaction with the sample. Note the similarity of the AFM Fig. Actually, the idea of AFM is based on the design of stylus profilometers, but the AFM can reveal the sample relief with subnanometer resolution. A set of styli probes the sample which is drawn below the set of styli.


    The small motions of the styli are transformed into an electrical signal by linear, variable transducers. Step heights of down to a few 10 nm are resolvable The force of tip-sample interaction Fig. That is why the cantilever must have a small mass, and the 7. Three support posts in the design of Fig. The xyz-translation stages for coarse adjustment of the piezoelectric scanner with the sample on top are engineered for little wobbling as well. The whole AFM is placed on a rubber support preventing transmission of high frequency vibrations from the laboratory not shown.

    A low force of interaction is crucial for high resolution force microscopy on soft biological specimens. Low spring constants of the cantilever may facilitate this purpose at the expense of resolution, but the most common way of gentle measurement is to reduce the intensity and duration of contact by oscillating the cantilever, as will be explained later Fig.

    The sample spot of interest is positioned near the tip by coarse xyz-translation stages. The piezoelectric scanner see also Fig. It allows motion control of the tip with subnanometer precision. A photodiode detects the reflection changes of a laser beam from the cantilever upon approach of tip to sample. In this example, the cantilever is mounted to a dithering piezo element which excites oscillations of the cantilever.

    The lock-in amplifier detects changes of these oscillations due to tipsample interactions. The sample surface is scanned by sample movement in horizontal direction by the piezoelectric scanner. The scanner also adjusts the relative height of the cantilever during scanning to avoid crashes of the tip with the sample surface. Such crashes can damage the tip and then cause artifacts see Fig. A voltage change at the x- or y- electrodes causes bending in the horizontal plane; contraction and expansion are generated by simultaneous application of x- and y-voltage Fig.

    Blunt tips and broken tips give rise to an seemingly flattened sample relief which may be difficult to recognize as an artifact 7 Scanning probe microscopy Fig. This self-made tip may also be used for STMs see Sect. One can make tips themselve by grinding a tungsten wire on a sheet covered with alumina Fig.


    These tips are also suitable for STM Sect. SiN is extremely hard, and tips can be engineered with radii of only a few nm Silicon nitride tips are the currently available tips with highest robustness Fig. Sharper tips with quite reproducible shape are made from silicon which is relatively fragile, however Fig. Even sharper tips for application on samples with particularly deep structures are manufactured by attaching a high 7. Sharp cantilevers for the examination of very rough surfaces Fig.

    High aspect-ratio tips made from silicon or carbon fiber are generally less durable than low aspect-ratio tips made from silicon nitride. In the latter mode, oscillations are excited by a piezo crystal and the forces between tip and sample are very small. This mode permits truly atomic resolution Giessibl, 7. In the contact mode, the probe tip is in continuous contact with the sample surface. The force the cantilever exerts on the substrate in contact mode may perturb the surface of soft biological materials.

    In the gentler dynamic mode, the probe tip only oscillates up and down as it is scanned over the sample surface. Two sub-modes may be distinguished for the dynamic force mode, the non-contact sub-mode in which the distance between tip apex and sample surface is always larger than the van-der-Waals distance, and the tapping sub-mode in which the tip has intermittent contact. As pointed out, a high degree of protection against external high-frequency vibrations is obviously crucial for the operation scanning probe microscopes with atomic resolution.

    Here the AFM is made from very thick and short plates of steel and the AFM is placed on three rubber balls that do not transmit fast vibrations. Another technique of efficient vibrational damping is to hang the AFM on a rubber string Fig. The vibrational damping is attained by a very rigid construction and an elastic support in form of three rubber balls 7 Scanning probe microscopy Fig. The direct visualization of a DNA glycosylase searching for damage shows that the glycosylase interrogates DNA at undamaged sites by introducing drastic kinks Chen et al.

    Intramolecular triplex DNA formation results in a kink in the double helix path Tiner et al. A sharp DNA bend is induced by binding of integration host factor IHF to the region between the upstream regulatory sequence and the promoter sequence Seong et al. Single DNA molecule force spectroscopy can discriminate between different interaction modes of small drug molecules with DNA by measuring the mechanical properties of DNA and their modulation upon binding of small drug molecules Krautbauer et al.

    A decrease of the ionic strength from 50 mM to 1 mM resulted in a change of the number of nodes crossings of double helical segments of a supercoiled bp piece of DNA from a 15 to one or two nodes Cherny and Jovin, AFM technology was used to map out the electrostatic potential of the transmembrane channel OmpF porin Fig. AFMs gave crucial topological information of blood cell adhesion on different sensor materials Hildebrand et al. Atomic force microscopy resolved fusion pores in the apical plasma membrane in live pancreatic cells Cho et al.

    Cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy.

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    • Atomic force microscopy allowed to visualize the structure of biomolecules, e. It is also an excellent technique to study the initial events of mutual cell adhesion Razatos, An AFM image of a monomolecular film of bovine serum albumin shows individual monomers and dimers Fig. Different apparent shapes of the porin are observed at different ionic strengths.

      These differences reflect changes of the electrostatic potential which is experienced by the charged tip of the AFM 7 Scanning probe microscopy Fig. Individual monomers and dimers of BSA can be seen AFMs are also very useful for the manipulation of macromolecules: proteins may physisorb to the AFM tip and can then be lifted and manipulated Fig. The sensitivity of the AFM cantilever, to forces in the pN range, was exploited to measure folding-unfolding forces within single protein molecules and breakaway forces between different biomolecules Jiao et al.

      Atomic force microscopy has yielded tantalizing insights into the dynamics of protein self-assembly and the mechanisms of protein unfolding Furuike et al. For further, similar applications of AFM technology see Chap. Due to the attractive action of the van-der-Waals interaction, the macromolecule sticks to the tip and can be lifted and moved to a different place 7. It was the first member of the family of scanning probe microscopes SPM that can characterize surface morphology with atomic resolution.

      In contrast to the AFM, its principle of operation Fig. A sharp tip located on a flexible cantilever is used to probe the distance between the tip and sample surface, as judged by the tunneling current Fig. Since the tunneling current also depends on the chemical nature of sample and tip, the STM also serves for characterization of electronic properties of solid samples.

      Significant complications on the way towards higher resolution of proteins are the undesired excitation of the soft biological material by the high current of STMs, typically pA— nA, and the distribution of conductivity within the sample distorting the image Fig. Low currents and stable attachment of the sample to the support are required for high resolution images of biological macromolecules. A finely sharpened electrically conductive tip is first positioned within about 1 nm of the sample by mechanical translation stages not shown and the piezoelectric scanner.

      At this small separation, electrons tunnel through the gap between tip and sample Fig. The tunneling current depends on the applied bias voltage between tip and sample, the distance, the tip shape, and the chemical compositions of sample and tip. The feedback loop ensures constant height or constant current. Tunneling current and feedback voltage are a measure of surface morphology and composition Binnig et al.

      Scanning tunneling microscopy demonstrated that the otherwise nearly linear mammalian metallothionein-2 molecule bends by about 20 degrees at its central hinge region between the domains in the presence of ATP Maret et al. Electrochemical scanning tunneling microscopy on thiolderivatized DNA immobilized on a gold single crystal surface showed 7.

      A STM study of morphology and electron transport features in cytochrome c offers evidence for sequential discrete electrontunneling effects Khomutov et al. Tunneling in proteins adsorbed onto a conductive substrate may depend on the applied potential Facci et al. The ability to site-specifically introduce cysteine residues and to engineer tags, such as histidine tags and biotin-acceptor peptides, allow the creation of ordered immobilized protein structures that can be characterized both electrochemically and topographically by using scanning probe microscopy and cyclic voltammetry Gilardi et al.

      By means of this technological innovation they achieve a resolution which may be well beyond the resolution limit, d, of classical Abbe-Fourier optics see also Sect. Using UV light and image processing can 7 Scanning probe microscopy yield improvement beyond this, but it is clear from Eq. SNOMs have been the first optical microscopes that significantly overcame the limit of Eq.

      An optical probe emits light from points above the 2D sample. The subwavelength probe tip takes advantage of the physical effect of optical tunneling. In this SNOM it is made using a metal coated tapered glass fiber: a first taper for the probe was manufactured by melt-drawing, and a second taper at the very end of the probe was etched.

      The damping of the horizontally vibrating SNOM probe caused by shear forces is taken as a measure for the distance from the sample.