HISTORICAL REVIEW

1911–1913

Otto Heimstaedt and Heinrich Lehmann built the first fluorescence microscopes in Germany in 1911. These microscopes were initially used to observe auto-fluorescence in living cells, but shortly after Stanislav Von Provazek applied fluorescence microscopy to the study of stained biological samples.

1931

M. Goppert-Mayer gave the theoretical background of two-photon excitation in fluorescence. Thirty years later, this phenomenon was observed experimentally shortly after the invention of the laser by W. Kaiser and C. Garrett. In 1990, W. Denk introduced two-photon excitation in fluorescent microscopy.

1941

Albert Coons described the fluorescent method of labeling antibodies, thus bringing forth the field of immunofluorescence.

1948

T. Forster formulated the principle of fluorescence resonance energy transfer (FRET), a phenomenon that occurs when two different chromophores (donor and acceptor) with overlapping emission/absorption spectra are separated by a suitable orientation and a distance in the range 20–100 Å. In the early 1970s, after a long period of inaction, the ground-breaking work of L. Stryer and R. P Hohland on FRET revealed the spatial proximity relationships of two fluorescence-labeled sites in biological macromolecules, thereby establishing FRET as a spectroscopic ruler. All of this early work used either fluorescent analogues of biomolecules or fluorescent reagents covalently or non-covalently attached to macromolecules as donors or acceptors of FRET. In the 1990s the introduction of the green fluorescent protein (GFP) to FRET-based imaging microscopy gave new life to its use as a sensitive probe of protein–protein interactions and protein conformational changes in vivo.

1961

B. Rotman was the first to use fluorescence detection for single-molecule studies in solution. Using a fluorogenic substrate, he measured the presence of a single β-D-galactosidase molecule by detecting the fluorescent product molecules accumulated in a microdroplet through enzymatic amplification. He did not achieve single-molecule sensitivity but clearly demonstrated the great potential of fluorescence detection. In 1976, T. Hirschfeld reported the use of fluorescence microscopy to detect single antibody molecules tagged with 80–100 fluorescein molecules under evanescent-wave excitation.

1964

S. Singh and L. Bradely predicted a three-photon absorption mechanism. In 1995 three-photon excited fluorescence spectroscopy and microscopy was demonstrated by a few groups and applied to the imaging of biological specimens and live cells.

The first lifetime measurements on single points under a microscope were carried out by a few groups in the early 1970s.

HISTORICAL REVIEW

1926

E. Gaviola created the first instrument for fluorescence measurement, which he called a fluorometer.

1934

F. Perrin formulated the relation between fluorescence depolarization and Brownian rotary motion for hydrodynamic spherical molecules. In 1936 he extended his treatment to symmetric top molecules.

1952

G. Weber first applied Perrin theory to biological macromolecules. Numerous fluorescence polarization measurements were carried out under steady-state conditions, utilizing constant illumination. Most of them were performed on dye-conjugated macromolecules to determine the harmonic mean rotational relaxation time. Weber's classical review of the application of polarized fluorescence, particularly to proteins, appeared in the 1950s.

1961

A. Jublonski proposed following the rotational relaxation process directly by measuring the decay of the fluorescence polarization as a function of time. He stressed that time-dependent anisotropy can be interpreted more directly and definitely than its time-average value observed with constant illumination.

1966

P. Wahl, and two years later L. Stryer, experimentally measured the time decay of fluorescence polarization on dye-conjugated globular proteins.

1969

T. Tao derived relations between the time-dependent fluorescence polarization anisotropy and the Brownian rotational diffusion coefficients of macromolecules. It was shown that in the most general case of a completely asymmetric body, five exponentials appear in anisotropy. In 1972 analogous results were obtained by other groups (G. G. Belford, R. L. Belford, and G. Weber; T. J. Chuang and K. B. Eisenthal).

Mid-1990s

A few groups reported the synthesis and spectral properties of long-lifetime, highly luminescent, metal–ligand complexes (B. A. De Graff and J. N. Demas, J. R. Lakowicz's group). These very photostable probes with microsecond decay times allowed measurements of rotational correlation times as long as 2 μs and detection of high-molecular-mass analytes in fluorescence polarization immunoassays.

1980–1995

J. R. Lakowicz's group made an essential contribution to the theory and practice of depolarized fluorescence spectroscopy. In 1987 the textbook Principles of Fluorescence Spectroscopy, by J. R. Lakowicz, appeared.

HISTORICAL OVERVIEW AND BIOLOGICAL APPLICATIONS

The term thermodynamics is derived from the Greek therme, meaning heat, and dynamis, meaning strength or force. Thermodynamics, as a science, has its beginnings in the nineteenth century, with the first experiments exploring the relationship between heat and work, the definitions of the concepts of temperature and energy and the clear enunciation of the first and second laws of thermodynamics. Classical thermodynamics uses a phenomenological approach based upon these laws, in contrast to statistical thermodynamics, which tries to establish a more fundamental understanding of heat in terms of the kinetics of large assemblies of atoms or molecules. The concept of work was generalized beyond mechanical work to include all forms of energy such as electric, magnetic, chemical, and radiation energy. The validity of thermodynamics was established for all types of system, from a volume of perfect gas at a given pressure to thermonuclear plasma, encompassing magnetic systems, liquid–vapor systems, macromolecular solutions, chemical reactions, etc. In this chapter we are concerned, in particular, with the applications of both classical and statistical equilibrium thermodynamics to biological macromolecules, their solutions and interactions.

Early in the seventeenth century, Galileo Galilei and his followers constructed the first thermometers, allowing reproducible measurements of temperature. Joseph Black (1728–1799) built the first calorimeter; he is considered the founder of calorimetry. His measurements led to the theory of caloric, a conserved elastic fluid that had weight and was associated with temperature. Antoine-Laurent Lavoisier (1780) performed the first calorimetric measurements in biology. He studied energy exchange in animals (even though the concept of energy had not yet been enunciated) by placing a guinea pig in a more elaborate ice calorimeter (developed with P. S. Laplace) than that of Black. The heat output was measured by the rate at which the ice melted; heat input was calculated by subtracting measurements on feces and urine from measurements on food intake. Thomas Young (1807) introduced the term energy (from the Greek en meaning “in” and ergon meaning “work”) but the distinction between force and energy was not made clearly at the time.

HISTORICAL REVIEW AND INTRODUCTION TO BIOLOGICAL PROBLEMS

Optical activity is the property of certain materials to change the angle of polarization of a beam of light that is shone through them as a function of its wavelength (optical rotation dispersion, ORD), or to absorb light differently according to its wavelength and polarization (circular dichroïsm, CD, from the Greek meaning related to two colors).

1812–1838

Jean-Baptiste Biot and Auguste Fresnel independently made careful studies of optical activity. Biot's polarimeter (an optical instrument used to measure the angle of rotation of polarized light) used sunlight, plane-polarized by reflection off glass, as a light source. Biot defined [α] the angle of specific rotation of the axis of plane-polarized light by an optically active solution in terms of the concentration in grams per cubic centimeter and the optical path in decimeters, a definition still in use. In 1824, Fresnel predicted that helical structures would be optically active.

1848

Louis Pasteur observed that optically inactive crystals of sodium ammonium tartrate were in fact mixtures of two classes. When separated, both classes turned out to be optically active with respective specific rotation angles of equal value but opposite in sign. The molecules making up the crystals were themselves in one of two asymmetric forms, enantiomorphs (from the Greek for opposite shapes), each a mirror image of the other. Since a two-dimensional molecule and its mirror image can be made to coincide and be considered to be identical, the implication was that molecules are three-dimensional (Comment E4.1). The form that turns the axis of polarized light to the right when viewed toward the light source in a polarimeter is called dextro-rotatory or the D-form; the form that turns the axis to the left is called the L-form or laevo-rotatory (from the Latin for right and left). The same terms apply to circularly polarized light (see Chapter A3); the electric vector of right-circularly polarized light turns in the clockwise direction when viewed toward the light source, the vector of left-circularly polarized light turns anticlockwise. Above a certain temperature (26 °C for sodium ammonium tartrate) the D- and L-forms mix to make up homogeneous crystals. They are said to form a racemate or racemic mixture (from the Latin, racemes, a bunch of grapes (optically inactive tartaric acid was obtained from grape juice)).

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.