HISTORY AND INTRODUCTION TO BIOLOGICAL PROBLEMS

Traditionally, hydrodynamics deals with the behavior of bodies in fluids and, in particular, with phenomena in which a force acts on a particle in a viscous solution. Very eminent scientists, such as Isaac Newton, James Clerk Maxwell, Lord Rayleigh (J. W. Strutt), and Albert Einstein, started their careers with major contributions to the science of hydrodynamics. Note that not only are the discoveries from more than 100 years ago still highly relevant today, but also that they continue to stimulate important new developments in the field.

1731

The science of hydrodynamics arose from the book Hydrodynamics by Daniel Bernoulli, which contained the “Bernoulli law” relating pressure and velocity in an incompressible fluid.

1821

Botanist Robert Brown described the random, thermal motions of small plant particles suspended in water, a phenomenon that was later named Brownian motion. In 1855 Adolf E. Fick published a phenomenological description of translational diffusion and deduced the fundamental laws governing transport phenomena in solutions. In the 1990s, the method of video-enhanced microscopy was proposed for the direct observation of Brownian motion of labeled macromolecules in a membrane.

1845

G. Stokes showed that the translational friction for a sphere is proportional to its radius and to the viscosity of its surrounding solvent. In 1856 he demonstrated that, for small angular velocity, the rotation of the sphere is characterized by a single parameter, which is proportional to the cube of the linear dimension. The way was then clear for a direct determination of particle dimensions from hydrodynamic measurements. In 1880, Stokes analyzed rotational friction and deduced an expression to relate the rotational friction coefficient of a particle to its volume.

1850

T. Graham observed that egg albumin diffuses much more slowly than common compounds such as salt or sugar. In 1861 he used dialysis to separate mixtures of slowly and rapidly diffusing solutes, and made quantitative measurements of diffusion on many substances. On the basis of this work he classified matter in terms of colloids and crystalloids.

1879

Publication of Hydrodynamics by Sir Horace Lamb in 1879.

André Guinier, whose fundamental discoveries contributed to the X-ray diffraction methods that are the basis of modern structural molecular biology, died in Paris at the beginning of July 2000, only a few weeks after it was announced in the press that a human genome had been sequenced. The sad coincidence serves as a reminder of the intimate connection between physical methods and progress in biology. Not long after, Max Perutz, Francis Crick and then David Blow, the youngest of the early protein crystallographers, passed away. The period marked the gradual closing of the era in which molecular biology was born and the opening of a new era. In what has been called the post-genome sequencing era, physical methods are now increasingly being called upon to play an essential role for the understanding of biological function at the molecular and cellular levels.

Molecular biophysics classical text books published in the previous decades have been overtaken not only by significant developments in existing methods such as those brought about by the advent of synchrotrons for X-ray crystallography or higher magnetic fields in NMR, but also by totally new methods with respect to biological applications, such as mass spectrometry and single molecule detection and manipulation. Our ambition in attempting this book was to be as up-to-date and exhaustive as possible. In their respective parts, we covered classical and advanced methods based on mass spectrometry, thermodynamics, hydrodynamics, spectroscopy, microscopy, radiation scattering, electron microscopy, molecular dynamics and NMR. But rapid progress in the field (we couldn't very well ask the biophysics community to stop working during the few years it takes to write and prepare a book!), and the requirement to keep the book to a manageable size meant that certain methods are either omitted or not perfectly up-to-date.

The key-word in molecular biophysics is complementarity. The Indian story of the six blind men and the elephant (see Frontispiece) is an appropriate metaphor for the field. Each of the blind men touched a different part of the elephant, and concluded on its nature: a big snake said the man who touched the trunk, the tusks were spears, its side a great wall, the tail a paint brush, the ears huge fans, the legs were tree trunks.

HISTORICAL REVIEW

Early 1980s

G. Binning and H. Rohrer proposed the scanning tunneling microscope (STM)(for which they were awarded the Nobel Prize). This invention has initiated an exciting series of novel experiments to image the surface of conducting as well as insulating solids with atomic resolution. The first attempts at imaging biological molecules using a STM date back to 1983. In 1987, individual molecules of phthalocyanine, lipid bilayers, and ascorbic acid were reported. One year later, H. Ohtani and collaborators imaged benzene, the molecule of Kekulé's blue dream, as three-lobed rings. In 1989 a spectacular view of the double-stranded Z-DNA molecule, the first biological macromolecule studied using an STM, was presented.

1986

A. Ashkin and coworkers proposed the idea of an optical trap (tweezers). An optical trap can be produced with a highly focused laser light, and can be used to grab, move, and apply measurable forces on micrometer-sized objects, such as dielectric microspheres. A microsphere that is chemically coupled to a molecule of interest provides a means of measuring the molecule's position and the force that it exerts.

G. Binning, C. F. Quate and C. Gerber invented the scanning force microscope (SFM). In this microscope a sensor tip carried by a flexible cantilever is used to touch and characterize a surface. This was a significant breakthrough; it allows biological macromolecules to be scanned in aqueous solution and gives reproducible imaging of DNA and of membrane protein crystals.

1990s

K. Bustamante and coworkers pioneered direct mechanical measurements of the elasticity of single DNA molecules using magnetic trapping. In 1994, S. Chu and coworkers studied the relaxation properties of single DNA molecules using optical trapping, and G. Li and colleagues made a direct measurement of the force between complementary strands of DNA with atomic force microscopy. Mechanical properties of individual strands and double-stranded DNA have been determined. In 1997, G. Shivashankar and A. Libchaber developed a new technique for single DNA molecule grafting and manipulation using combined atomic force microscopy and an optical tweezer. These studies opened up new possibilities in biosensors and bioelectronic devices. K. Svoboda and coworkers applied optical trapping nanometry (optical tweezers in conjunction with nanometer-precision position detection schemes) to study a single kinesin molecule.

HISTORICAL REVIEW

Ultrasound refers to sound waves of frequencies higher than the hearing capacity of the human ear (between 20 Hz and 20 kHz). Frequencies used in medicine are usually greater than 2.5 MHz. Issuing from military and industrial applications for SONAR (sound navigation and ranging) and metal flaw detection, ultrasonic energy to “view” inside the human body was developed in parallel in hospitals of many countries around the world, including the USA, Europe, Japan, China, and Australia. The brief history below summarizes the main developments in the USA and Europe.

Ultrasound emerged in the 1940s as a diagnostic, therapeutic, and then surgical tool using high intensities to break down tissue. The first diagnostic application is accepted to be due to Karl Theodore Dussik and collaborators in Austria. The work described in a paper published in 1942 showed images claimed to be of cerebral ventricles and included a discussion that low-intensity ultrasound could be used to locate brain tumors. Dussik and his collaborators had used a transmission method and the published images were later criticized as being artifacts due to reflections of sound waves within the skull. The transmission method of ultrasound imaging was subsequently dropped.

In the USA, George Ludwig applied ultrasound to systematic imaging of various tissues and organs from animals and measured the sound impedance of different types of gallstone, muscle, and fat, in the body. He was a naval officer and his work was classified and only released for publication in 1949. At about the same time, John Wild, a surgeon, used the one-dimensional (A-mode) ultrasound investigation method to assess the thickness of bowel tissue under different surgical conditions. Then, with John Reid, a recent graduate in electrical engineering, they built a B-mode (two-dimensional) instrument able to visualize tumors by scanning breast lumps.

In 1953 in Sweden, Carl Helmut Hertz, a graduate student in physics (son of Gustav Ludwig Hertz in honor of whom the unit of frequency, in s–1, was named) collaborated with cardiologist Inge Edler to make the first successful ultrasound measurement of heart activity, and generated the first echo-encephalogram (ultrasound brain scan). C. H. Hertz was already familiar with the application of ultrasound to non-destructive materials testing, and they made the measurements on an instrument borrowed from a ship construction company.

The present chapter is included in Part E with the other optical spectroscopy methods; however, the development of two-dimensional infrared (2D-IR) spectroscopy is strongly based on two-dimensional NMR, and it is easier to understand after reading the relevant sections in Part J, which the reader is strongly encouraged to do first.

HISTORICAL REVIEW AND INTRODUCTION TO BIOLOGICAL PROBLEMS

1950

O. Hann proposed coherent spectroscopy – the use of radiation fields with well-defined phase properties – to extract information about atoms and molecules. The “spin echo” experiment in nuclear magnetic resonance was the first demonstration of the possibilities of coherent spectroscopy.

1957

R. P. Feynman, F. L. Vernon Jr., and R. W. Hellwarth published a landmark paper pointing out that if coherent light fields were ever created, it would be possible to use these same methods on optical transitions. The invention of the laser in 1960 was followed quickly by a demonstration of the “photon echo” – the optical version of the “spin echo.”

1998

R. M. Hochstrasser and collaborators proposed 2D-IR spectroscopy, in analogy with two-dimensional NMR, for the determination of time-evolving structures. The spins associated with the different nuclei in NMR are replaced in the IR experiments by a network of vibrational modes whose coupling can be used to determine molecular structure and dynamics. Structures of dipeptides, tripeptides, and pentapeptides were determined by 2D-IR spectroscopy. The most exciting aspect of 2D-IR spectroscopy, however, is the combination of its sensitivity to structure with time resolution.

Conventional FTIR spectroscopy can monitor protein secondary structure features on the nanosecond–second timescale. Pulsed IR methods can probe vibrations on timescales down to the femtosecond.

LINEAR AND MULTIDIMENSIONAL SPECTROSCOPY

Linear spectroscopy, such as visible or IR absorption, Raman scattering, and one-dimensional NMR, produces one-dimensional projections of electronic and nuclear interactions onto a single frequency (or time) axis. For simple molecules, direct information can be obtained about energy levels and absorption cross-sections. The situation is very different for complex molecules, with strongly overlapping levels. Here, the microscopic information is highly averaged, and is often totally buried under broad, featureless line shapes, whose precise interpretation often remains a mystery.

HISTORICAL REVIEW

In 1963 Richard Feynman traced to Pythagoras (c.500 BC) the first example, outside geometry, of the discovery of a numerical relationship in Nature. Pythagoras’ discovery, which led to the foundation of a school of thought with mystic beliefs in the power of numbers, was that two strings under the same tension but of different lengths give a pleasant sound when plucked together if the ratio of their lengths is that of two small integers. We now say that a ratio of 1:2 corresponds to an octave, a ratio of 2:3 to a fifth, and so on, which are all harmonic-sounding chords. Feynman analyzed this discovery in terms of three characteristics: its basis in experimental observation; the use of mathematics as a tool for understanding Nature; and its concern with aesthetics (the “pleasant” quality of the sound). With easy hindsight (as he readily admits!) Feynman wrote that if Pythagoras had been more impressed by the first point on the importance of experimental observation the science of physics might have had an earlier start.

We discovered the existence and seek our biophysical understanding of macromolecules through experimentation, and we use mathematical tools not only to set up experiments and analyze the results, but also for the description of the studied “objects” themselves. The basis of aesthetics may still remain as mysterious as in Pythagoras’ time, but there is no doubt that the “beauty” of the DNA double helix and the satisfyingly elegant way in which it provided an explanation for how genetic information is stored and transmitted was an essential inspiration in the development of modern molecular biology. Similarly, the usual, colorful illustrations of protein structural models are undoubtedly aesthetic and certainly play a role in the acceptance and understanding of these models by biologists, who might have been put off by a purely mathematical interpretation of the data, even if it were more accurate.

Experiments on biological macromolecules are difficult to perform and interpret. Sample material is fragile and a good biophysical experiment must rest on a firm biochemical foundation. Biological macromolecules are much smaller than the wavelength of light and cannot be “seen.”

The ideal biophysical method would be capable of measuring atomic positions in molecules in vivo. It would also permit visualization of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the timescale involved. At present there is no single experimental technique that can yield this information.

A BRIEF HISTORY AND PERSPECTIVES

Molecular biology was born with the double-helix model for DNA, which provided a superbly elegant explanation for the storage and transmission mechanisms of genetic information (Fig. 1). The model by J. D. Watson and F. H. C. Crick and supporting fiber diffraction studies by M. H. F. Wilkins, A. R. Stokes, and H. R Wilson, and R. Franklin and R. G. Gosling, were published in a series of papers in the April 25, 1953 issue of Nature, and marked a major triumph of the physical approach to biology.

The Watson and Crick model was based only in part on data from X-ray fiber diffraction diagrams. The patterns, which demonstrated the presence of a helical structure of constant pitch and diameter, could not provide unequivocal proof for a more precise structural model. One of the “genius” aspects of the discovery was the realization that A–T and G–C base pairs have identical dimensions; as the rungs of the double-helix ladder, they give rise to a constant diameter and pitch. From a purely “diffraction physics” point of view, a variety of helical models was compatible with the fiber diffraction diagram, and other authors proposed an alternative model for DNA, the so-called “side-by-side model,” coupling two single DNA helices. This shows that if molecular biology were ever to be established, it was important to obtain the structure of biological molecules in more detail than was possible from fiber diffraction. Considering the dimensions involved, about 1 Å (0.1 nm) for the distance between atoms, X-ray crystallography appeared to be the only suitable method. Major obstacles remained to be overcome, such as obtaining suitable crystals, coping with the large quantity of data required to describe the positions of all the atoms in a macromolecule, and solving the phase problem.

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)).