HISTORICAL REVIEW

T. Wiseman, S. Williston, J. F. Brandts, and L. N. Lin published a paper in 1989 with the title: “Rapid measurement of binding constants and heats of binding using a new titration calorimeter.” The term isothermal titration calorimetry (ITC) was introduced by E. Freire and colleagues in 1990. The method is unique in providing not only the magnitude of the enthalpy change upon binding, but also, in favorable experimental conditions, values for the binding affinity and entropy changes. Because these parameters fully define the energetics of the binding process, ITC is playing an increasingly important role in the detailed study of protein–ligand interactions and the associated molecular design approaches, in particular with respect to drug design. In the 1990s, a number of critical reviews of ITC results and analytical developments were published.

EXPERIMENTAL ASPECTS AND EQUATIONS

Measuring Protocol and Samples

The reaction cell in ITC has a volume close to 1 ml and contains one of the reactants. The other reactant is added to it by injection in small volumes (close to 10 μl), and stirred in. The amount of power (in millijoules per second or in watts) required to maintain a constant temperature difference between the reaction bath and a reference cell is measured by the calorimeter. The heat absorbed or released by the chemical reaction is determined from the integral of the power curve over the appropriate time (Fig. C3.1).

In the ITC experiment of Fig. C3.1, the 0.6 ml calorimeter cell was filled with protein solution (68.8 mM pancreatic RNase A), and the ligand solution (0.5 mM cyclic monophosphate) was added in precise 5 μl volumes through an injection syringe that also stirred the resulting solution to ensure proper mixing. The quantity plotted on the y-axis is the time dependence of the power needed to maintain a constant temperature difference between the measuring and a reference cell. Each peak corresponds to one injection, and its integral (after subtraction of the background due to ligand dilution, mechanical mixing effects etc.) to the heat associated with the amount of binding (qj in Eq. (C3.1)).

The peaks are progressively smaller with each injection because there is less free protein available for binding. When the protein is saturated, the peak height represents the background level. The integral heat effect of the binding as a function of injection number is plotted in Fig. C3.1b.

BRIEF HISTORICAL REVIEW AND BIOLOGICAL APPLICATIONS

1704

In Opticks Isaac Newton dealt with the formation of a spectrum by a prism, and the composition of white light and its dispersion. The Latin word, spectrum, means an appearance; a spectrum is obtained when radiation is broken up into its color or wavelength distribution.

1800

The astronomer William Herschel discovered infrared (IR) radiation.

1801

The physicist Johann Wilhelm Ritter discovered ultraviolet (UV) radiation.

1814

Joseph von Frauenhofer showed that the Sun's spectrum contained dark lines (later named Frauenhofer lines), indicating that light of the corresponding color was missing because of absorption.

1850–1900

August Beer stated the empirical law, which was named after him, that there is an exponential dependence between the transmission of light through a substance, the concentration of the substance, and the path length of the beam through it. The law is also known as the Beer–Lambert law or the Beer–Lambert–Bouguer law, in recognition of the work of Pierre Bouguer (1729) and Johann Heinrich Lambert (1760). Gustav Kirchhoff's discovery that each pure substance has a characteristic spectrum provided the basis for analytical spectroscopy. Gustav Kirchhoff and Robert Bunsen identified the chemical elements in the Sun by analyzing its spectrum. Johann Jacob Balmer identified a numerical series in the spectrum of hydrogen. Joseph John Thomson discovered the electron. Max Planck introduced the concept of quanta in the treatment of heat radiation and laid the foundation of quantum theory. He was awarded the Nobel Prize in 1918.

1911–1913

Ernest Rutherford proved that atoms comprise positively charged nuclei surrounded by electrons. Niels Bohr made the first theoretical calculations of the discrete energy states in the hydrogen atom and the related wavelengths of the emitted radiation lines.

Quantum and wave mechanics, which was developed in the 1920s by Erwin Schroedinger, Werner Karl Heisenberg, Wolfgang Pauli, and Paul Adrien Maurice Dirac, now provides a firm basis for the theoretical understanding and application of spectroscopic methods to study matter.

The first mid-IR spectrometer was constructed less than 35 years after the discovery of IR radiation, and within 90 years IR spectroscopy was finding applications in astronomy and organic and atmospheric chemistry.

HISTORICAL REVIEW OF BIOLOGICAL APPLICATIONS

The first molecular dynamics simulation of a biological macromolecule, the small stable protein bovine pancreatic trypsin inhibitor (BPTI) for which an accurate X-ray crystallographic structure was available, was published by J. A. McCammon, B. R. Gelin, and M. Karplus in 1975. Based on a simple approximation for the molecular mechanics potential, the simulation was performed for the macromolecule in vacuum, and limited to 9.2 ps by the computing power available at the time. The BPTI study was nevertheless effectively instrumental, together with the earlier hydrogen exchange experiments of K. Linderstrom-Lang and his collaborators (1955), in establishing the view that proteins are not rigid bodies but dynamic entities whose internal motions must play a role in their biological activity.

In 2003, M. Karplus wrote that molecular dynamics simulations of biological macromolecules developed from two lines of work: individual particle trajectory calculations in chemical reactions and physical calculations to predict the dynamic behavior of large particle assemblies.

The first line of work goes back to the two-body scattering problem for which analytical solutions are available. It is well known in physics, however, that no analytical solutions have been found for the three-body (or more) problem. A prototype trajectory calculation was attempted in 1936 by J. A. Hirschfelder, H. Eyring, and B. Topley for the simple chemical reaction involving the interconversion between hydrogen atoms and molecules, but the calculation could only be completed by using the computers that became available in the 1960s. Classical chemical trajectory calculations have now evolved to incorporate quantum mechanical effects.

The second line of work has its origins in the work of J. D. van der Waals on particle–particle interactions and the statistical mechanics of L. Boltzmann. In the 1950s, B. J. Alder and T. E. Wainwright published a study of liquids whose molecules behaved like “hard spheres.” A. Rahman in 1964 published a molecular dynamics simulation of liquid argon, assuming a “soft” Lennard–Jones potential; the first simulation of liquid water, a much more complex liquid, was published in 1971, by F. H. Stillinger and A. Rahman.

HISTORICAL REVIEW

1869

J. Tyndall performed the first experimental studies on light scattering from aerosols. He explained the blue color of the sky by the presence of dust in the atmosphere.

1871

Lord Rayleigh presented the theory of scattering from assemblies of non-interacting particles that were sufficiently small compared with the wavelength of light. According to Rayleigh, scattering by a gas occurs because of the fluctuation of the molecules around a position of equilibrium. Rayleigh obtained the formulae that explained the blue color of the sky as being due to the molecules in the atmosphere preferentially scattering blue light in comparison to red light.

1906

L. Mandelshtam raised the question on the nature of scattered light once again. He pointed out that Rayleigh's arguments do not fully explain the scattering phenomenon. According to Mandelshtam, light scattering is also due to the random fluctuations of molecules near the position of equilibrium. As a result of random fluctuations of order λ3, where λ is the scattering wavelength, the number of macromolecules will vary with time. It follows from the Mandelshtam theory that the translational and rotational diffusion coefficients of macromolecules could be obtained from the spectrum of the scattered light. In 1924, L. Mandelshtam described theoretically and experimentally what he termed combination light scattering, which later was renamed Raman scattering. As early as 1926 he recognized that the translational diffusion coefficient of macromolecules can be obtained from the spectrum of the light they scatter. However, at that time, lack of spatial coherence and monochromaticity in conventional light sources rendered such experiments impossible.

1914

L. Brillouin predicted a doublet in the frequency distribution of scattered light due to scattering from thermal sound waves in a solid. This doublet was later named after him. E. Gross performed a series of light-scattering experiments on liquids in 1932. He observed not only the Brillouin doublet but also a central peak, the position of which was unshifted. In 1933 L. Landau and G. Placzek gave a theoretical explanation of the central line from a thermodynamic point of view and calculated the ratio of the integrated intensity of the central line to that of the doublet.

1916

M. von Smoluchowski gave the first theoretical description of the amplitude and the temporal decay of number fluctuations in a diffusion system.

HISTORICAL REVIEW

Nobel Prizes

In 1901, the very first Nobel Prize in physics was awarded to W. C. Röntgen “in recognition of the extraordinary services he has rendered by the discovery of the remarkable rays subsequently named after him.” J. J. Thomson obtained the Nobel Prize in physics in 1906 “in recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases.” M. von Laue was awarded the physics Prize in 1914 “for his discovery of the diffraction of X-rays by crystals,” and W. H. Bragg and W. L. Bragg in 1915, “for their services in the analysis of crystal structure by means of X-rays.” J. Chadwick was awarded the Nobel Prize in physics in 1935 “for the discovery of the neutron.” L. Pauling was awarded the Nobel Prize in 1954 for his research into the nature of the chemical bond.

The birth of molecular biology was honored appropriately with the award of the 1962 Nobel Prize in physiology or medicine to J. Watson, M. Wilkins, and F. Crick “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material,” and, in the same year, the award of the chemistry prize to M. F. Perutz and J. C. Kendrew “for their studies of the structures of globular proteins.” D. C. Hodgkin, who had been recommended for the prize in the same year as Perutz and Kendrew, was awarded the 1964 prize in chemistry “for her determinations by X-ray techniques of the structures of important biochemical substances.” The 1982 Nobel Prize in chemistry was awarded to A. Klug “for his development of crystallographic electron microscopy and his structural elucidation of biologically important nucleic acid–protein complexes,” and the 1985 chemistry Prize was given to H. A. Hauptman and J. Karle “for their outstanding achievements in the development of direct methods for the determination of crystal structures.” The first high-resolution X-ray crystallography structure of a membrane protein complex was rewarded by the 1988 chemistry Prize to J. Deisenhofer, R. Huber, and H. Michel “for the determination of the three-dimensional structure of a photosynthetic reaction center.”

HISTORICAL REVIEW AND INTRODUCTION TO BIOLOGICAL APPLICATIONS

The wave nature of light, on which diffraction phenomena are based, was first suggested by Huygens more than 300 years ago. About 100 years later, Haüy wrote an essay on the regularity of crystal forms that is considered to be the beginning of crystallography.

1690

In his Treatise on Light, C. Huygens wrote that light “spreads by spherical waves, like the movement of Sound,” and explained reflection and refraction by wave constructions.

1784

R.-J. Haüy, a mineralogist, published his theory on crystal structure following observations that calcite cleaved along straight planes meeting at constant angles.

1895

J. J. Thomson discovered electrons during an investigation of cathode rays. He initially called them corpuscles.

1895

W. C. Röntgen discovered X-rays. While experimenting with electric current flow in a partially evacuated glass tube, he noted that radiation was emitted that affected photographic plates and caused a fluorescent substance across the room to emit light.

1912

P. P. Ewald's doctoral thesis on the passage of light waves through a crystal of scattering atoms led M. von Laue to ask what would happen if the wavelength of the light were similar to the atomic spacing, and this led to the first observations of X-ray crystal diffraction by W. Friedrich, P. Knipping, and von Laue. Because of their short wavelengths, X-rays provide a “ruler” with which to measure distances between atoms.

1912–1915

W. H. Bragg and W. L. Bragg interpreted diffraction in terms of reflection from crystal planes. They solved the crystal structures of NaCl and KCl and introduced Fourier analysis of the X-ray measurements.

1917

P. P. Ewald introduced the “reciprocal lattice” construction, a graphical method of expressing the geometrical conditions for crystal diffraction.

1924

W. L. Bragg and collaborators developed the use of absolute intensities in crystal analysis, leading to the solution of structures more complex than the monovalent salts.

As we wrote in the preface to the first edition, our ambition in attempting Methods in Molecular Biophysics was to be as up-to-date and exhaustive as possible, considering the rapid progress in the field. Judging by broad readers’ responses, the book usefully fulfilled its mission. The historical introduction to each method and “physicist” and “biologist” boxes were especially appreciated. Criticism focused on the inclusion of methods which even if once important are no longer topical, and relative inattention to emerging methods that were subsequently proven to be very powerful. Scientific predictions are, of course, particularly difficult to make, especially as progress may come from difficult to foreknow technical breakthroughs. The development of new detector systems, which now permit approaching atomic resolution in cryo-electron microscopy, comes to mind. The unwieldy size and weight of the first edition also invited justified criticism (it is interesting to note that the Russian edition is in two tomes). In this second edition, we have chosen a different book format that we hope will be easier and more pleasant to handle. We have carefully gone through the text to reorganize, bring up-to-date, and prune each of the chapters. We have added a new section on medical imaging so that the book now includes the range of topics covered in most medical school biophysics courses.

To the list of colleagues gratefully acknowledged in the first edition preface, we would like to add Frank Gabel, Institut de Biologie Structurale, Grenoble, for his critical reading of the first edition to suggest corrections and improvements, and expert colleagues who checked the updates, revisions, and additions in the second edition: Elisabetta Boeri Erba, Martin Blackledge, Dimitrios Skoufieas of the Institut de Biologie Structurale, Grenoble; Harriet Crawley-Snowdon, James Edgar, Antoni Wrobel, of the Cambridge Institute for Medical Research, University of Cambridge; Antony Fitzpatrick, Laboratory of Molecular Biology, Cambridge; Massimo Antognozzi, School of Physics, University of Bristol; Lotte Stubkjaer Fog, Medical Physicist, Section for Radiotherapy, Oncology Clinic, Rigshospitalet, Copenhagen; Alberto Bravin, Bio-medical Beam Line, European Synchrotron Radiation Facility, Grenoble; Jeremy Smith, Governor's Chair and Director, University of Tennessee/Oak Ridge National Laboratory Center for Molecular Biophysics.

HISTORICAL REVIEW AND BIOLOGICAL APPLICATIONS

Late 1600s

R. Boyle questioned the extremely practically oriented chemical theory of his day and taught that the proper task of chemistry was to determine the composition of substances.

1750

A.-L. Lavoisier studied oxidation, and correctly understood the process. He demonstrated the quantitative similarity between chemical oxidation and respiration in animals. He is considered the father of modern chemistry.

Late 1700s

The work of J. Priestley, J. Ingenhousz, and J. Senebier established that photosynthesis is essentially the reverse of respiration.

1800s

The development of organic chemistry, despite the strong opposition of vitalists (who believed that transformations of substances in living organisms did not obey the rules of chemistry or physics but those of a vital force), led to the birth of biochemistry. In 1828, F. Wöhler performed the first laboratory synthesis of an organic molecule, urea. During the 1840s, J. V. Liebig established a firm basis for the study of organic chemistry and described the great chemical cycles in Nature. In 1869 a substance isolated from the nuclei of pus cells was named nucleic acid. O. Avery's experiments of 1944 on the transformation of pneumococcus strongly suggested that nucleic acid was the support of genetic information.

1860s

L. Pasteur is considered the father of bacteriology. He proved that microorganisms caused fermentation, putrefaction, and infectious disease, and developed chemical methods for their study. In 1877 Pasteur's “ferments” were named enzymes (from the Greek en “in” and zyme “leaven or yeast.”) In 1897 E. Buchner showed that fermentation could occur in a yeast preparation devoid of living cells.

1882

E. Fischer showed that proteins are very large molecules built of amino acid units; he also discovered the phenomenon of stereoisomerism in carbohydrates.

1926

Urease, the first enzyme to be crystallized in a pure form, was shown to be a protein by J. B. Sumner. In the 1960s, M. Perutz and J. Kendrew solved the molecular structures of hemoglobin and myoglobin, respectively, by crystallography, thus proving that proteins had well-defined structures. They were awarded the Nobel Prize.