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 behaviour 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 (or more)-body 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.

History and introduction to bioliogical problems

Traditionally, hydrodynamics deals with the behaviour 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 classical book Hydrodynamics by Daniel Bernoulli, which contained the ‘Bernoulli law’ relating pressure and velocity in an incompressible fluid, as well as a number of its consequences. The next fundamental contribution to the field was in 1879 when Sir Horace Lamb published another classical book also named Hydrodynamics.

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 labelled macromolecules in a membrane.

1846

J. L. M. Poiseuille produced a theory of liquid flow in a capillary. Based on this theory, Wilhelm Ostwald invented the viscometer and introduced its use in physical and chemical experiments.

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 in the understanding of biological function at the molecular and cellular levels.

Classical molecular biophysics textbooks 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 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.

Historical overview and introduction to biological problems

1801

Thomas Young observed that two slits placed in front of a light source created a pattern of intensity fringes on a screen. This phenomenon is the basis of modern interferometers, which have now been adapted to measure ligand–receptor interactions.

1902

R. W. Wood observed for the first time the phenomenon of surface plasmon resonance (SPR), which provided a simple and direct sensing technique for probing refractive index changes that occur in the very close vicinity of a thin metal film surface.

1968

E. Kretschmann and H. Z. Raether proposed the basic configuration of an SPR sensor, with an optical system containing a prism coupled to a reaction cell.

1982–1983

B. Liedberg and colleagues realised the potential of SPR for macromolecular binding studies since the change in refractive index is dependent on the molecules accumulating at the metal surface. They adsorbed an antibody specific to immunoglobulin G onto a gold sensing film, resulting in the selective binding and detection of the protein.

1980s, 1990s

Several biosensors, based on either interferometry or SPR, that allowed the simple, rapid and non-labelled assay of various biochemical analytes such as proteins, DNA and small compounds, became commercially available.

1993

The potential to analyse weak affinities by SPR instrumentation was demonstrated by S. Davis and coworkers in their studies of cell surface receptor interactions.

2000 and after

Optical arrays were developed to analyse the interactions of thousands of different molecules simultaneously, with high selectivity and sensitivity.

Historical review

1916

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

1972–1974

D. Magde, E. L. Elson and W. W. Webb published a rigorous formalism of fluorescence correlation spectroscopy (FCS) with its various modes of possible application, highlighting the large potential of this variant of the relaxation method.

1990

R. Rigler and coworkers made the final breakthrough for the FCS method. They reached the single-molecule detection limit by combining FCS with a confocal set-up, thus increasing the signal-to-noise ratio dramatically. By tightly focusing a laser beam and inserting a pinhole into the image plane, maximum lateral and axial resolution were achieved.

1994

M. Eigen and R. Rigler triggered an important further development by proposing the application of dual-colour cross-correlation for diagnostic purposes. The underlying idea was to separate single-labelled reaction educts from dual-labelled reaction products to discriminate against an excess of free single-labelled species and thus enhance the specificity of detection. In 1997 P. Schwille and coworkers successfully monitored a hybridisation reaction of two differently labelled oligonucleotides by the dual-colour cross-correlation technique. In 2000 K. G. Heinze and coworkers, reported the application of dual-colour two-photon cross-correlation to determine enzymatic cleavage of a DNA substrate by endonuclease.

2000 to now

FCS has evolved into a whole family of related methods sharing the basic principle of fluorescence fluctuation analysis.

Historical review

The discoveries of X-rays, electrons and neutrons as major achievements in the field of biological crystallography have been honoured by Nobel prizes in physics, chemistry and physiology or medicine, spread over the more than 100 years since their inception 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 physics prize 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’.

The birth of molecular biology was honoured 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’.

Historical review

1846

J. L. Poiseuille produced a theory of liquid flow in a capillary. Based on this theory, W. Ostwald (1933) and L. Ubbelohde (1936) invented viscometers and introduced them into physical and chemical practice. Later, the instruments were named after them.

1906

A. Einstein was the first to treat the viscosity of suspensions of rigid spherical particles that are large relative to the size of the solvent molecules. He showed that the specific viscosity of the solution is proportional to the volume fraction of the solution occupied by the particles and does not depend on the absolute size of the spheres.

1932

H. Staudinger proposed using the intrinsic viscosity to determine the molecular mass of polymers. He found that the dependence of the intrinsic viscosity on the molecular mass for a homologous series of polymers can be expressed by a simple formula of the type [η] ≈ Mα. It became understood later (H. Mark, R. Houwink, W. Kuhn and H. Kuhn) that the constant α is related to the molecular conformation of the macromolecules in solution.

1962

B. H. Zimm and D. M. Crothers proposed an original design for a rotational viscometer that operates at low velocity gradients and which has been very useful for measurements on asymmetric structures such as DNA, fibrous proteins and rod-like viruses.

1985

M. A. Haney invented a ‘differential viscometer’ which measures the relative viscosity directly.

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 depolarisation 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 polarisation measurements were carried out under steady-state conditions, utilising 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 polarised 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 polarisation 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 polarisation on dye-conjugated globular proteins.

1969

T. Tao derived relations between the time-dependent fluorescence polarisation 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).

The ideal biophysical method would be capable of measuring atomic positions in molecules in vivo. It would also permit visualisation of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the time scale 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 fibre diffraction studies by M. H. F Wilkins, A. R. Stokes, and H. R Wilson, and R. Franklin and R. G. Gosling, published in a series of papers in the 25 April, 1953 issue of Nature, 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 fibre 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 realisation 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.

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 generalised 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–vapour 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.

Historical review

1920s

M. Smoluchowski and E.Hückel were the first to elaborate the theory of electrophoresis for thin and thick double layers. D. C. Henry provided a theory for the electrophoresis of spherical polyions that was valid for double layers of arbitrary thickness. In the Henry model, charge distribution within the sphere is assumed to be spherically symmetric, and ion relaxation is ignored. In the 1950s a number of investigators (J. Th. G. Oberbeek, F. Booth, P. H. Wiersma) studied the effect of ion relaxation on the electrophoretic mobility of spheres containing a centrosymmetric charge distribution.

1930

A. Tiselius presented a new technique for studying the electrophoretic properties of proteins. He showed that sharp electrophoretic moving boundaries of ionised molecules could be obtained in U-shaped quartz glass tubes and that the protein boundary could be detected with UV light. A. Tiselius described moving boundaries corresponding to albumin, α-, β-, and γ-globulin in serum.

1940s

T. Wieland and E. Fischer proposed the use of filter paper as a supporting medium for electrophoresis. Simplicity of construction, relative cheapness and compactness, and the requirement of much less than 1mg of protein for analytical or diagnostic purposes popularised paper electrophoresis.

1950s

O. Smithies obtained good resolution of proteins using starch gels. Electrophoresis with molecular sieving of a gel gave a much higher resolution than electrophoresis in free solution. In this decade, many materials (like strips of cellulose acetate, agarose) were introduced as supports for electrophoresis.

Brief historical review and biological applications

1704

In OpticksIsaac 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 colour or wavelength distribution.

1800

The astronomer, William Herschel, discovered infrared (IR) radiation.

1801

The physicist Johann Wilhelm Ritter discovered ultraviolet (UV) radia-tion.

1814

Joseph von Frauenhofer showed that the Sun's spectrum contained dark lines (later named Frauenhofer lines), indicating that light of the corresponding colour 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 transmision 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 analysing 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.

Theory of small-angle scattering from particles in solution

Small-angle scattering (SAS) is a very useful method in biochemistry, providing information on molecular masses, shapes and interactions in solution (see Comment G2.1).

Dilute solutions of identical particles

In order to have an observable signal in X-ray or neutron SAS a solution of the order of 100 μl containing a few milligrams per millilitre of macromolecule is required – corresponding to about 1015 particles for a 50 kDa protein at 1 mg ml−1 (we note that 1 mg ml−1, the usual unit in biochemistry is in fact equal to 1 g l−1, the unit more conveniently used in equations).

We first consider solution conditions such that the particles do not influence each other, i.e. the position and orientation of each particle is totally independent of that of the others. This is the infinite dilution condition, for which we say there is no interparticle interference. In practice, it is achieved at different, low, concentrations for different macromolecules and solvents. For example, the condition may well be satisfied for a given protein at a few milligrams per millilitre, in a neutral pH buffer, but tRNA molecules, which are highly charged at pH 7 in low-salt buffer, interact with each other even at these concentrations, and it might not be possible to reach the infinite dilution condition without increasing the solvent salt concentration.

Historical review

J.-B. Biot and F. Savart collaborated on the theory of magnetism. Their eponymous law (formulated in 1820) describes the motion of a charge in a magnetic field.

Ernst Abbe in 1872 formulated his wave theory of microscopic imaging. In 1878, he was able to correlate resolution and wavelength of light mathematically. A few years later, through the company Zeiss, he commercialised the first ever lenses to have been designed based on sound optical theory and the laws of physics.

In the second half of the nineteenth century, J. C. Maxwell established the laws of electromagnetism. His equations first appeared in fully developed form in his book Electricity and Magnetism (1873).

In 1897 J. J. Thomson discovered electrons by demonstrating cathode rays were actually units of electrical current made up of negatively charged particles of subatomic size. For these investigations he won the Nobel Prize for physics in 1906.

In his doctoral thesis of 1924, de Broglie theorised that matter has the properties of both particles and waves. He received the 1929 Nobel Prize for physics. The particle–wave duality was confirmed experimentally, for the electron, a few years later by C. J. Davisson and L. H. Germer and by G. P. Thomson. Thomson and Davisson jointly shared the Nobel Prize in physics in 1937.

In 1926 H. Busch described a lens for electrons. H. Ruska subsequently built the first working electron microscope in 1930 (Nobel Prize in 1986).

Historical review

Early 1980s

G. Binning and H. Rohrer proposed the scanning tunnelling microscope (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 scanning tunnelling microscope (STM) date back to 1983. In 1987, individual molecules of phthalocyanine, lipid bilayers and ascorbic acid were reported. One year later, H. Ohtani with collaborators imaged benzene, the molecule of Kekul′e '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 a STM, was presented.

1986

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 characterise a surface. This was a significant breakthrough which allows biological macromolecules to be scanned in aqueous solution and gives reproducible imaging of DNA and of membrane protein crystals.

Early 1990s

D. J. Keller, Q. Zhong and C. A. J. Putman independently proposed the so-called tapping mode of the atomic force microscope (AFM), in which the cantilever is oscillated vertically while it is scanned over the sample. In this mode the image is formed by displaying the reduction of the oscillation amplitude at every point of the sample.

Historical review

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.

1855

A. Fick, in the equations now known as his first and second laws, defined a diffusion coefficient (D) to describe the flow of a solute down its concentration gradient. J. Stefan (1879) and L. Boltzmann (1894) developed mathematical integral forms of the second law, opening the way for the experimental determination of diffusion coefficients by various methods.

1905–1906

A. Einstein, W. Sutherland and M. von Smoluchowski determined that steady-state solutions of Fick's equations have a direct analogy in heat flow along a temperature gradient, and established mathematical relations between diffusion and frictional coefficients in Brownian motion. In 1905, W. Sutherland used a value of D calculated by J. Stefan from data collected by T. Graham, to obtain a molecular weight of 33000 for egg albumin (the true value is about 45000).

1926

L. Mandelshtam recognised that the translational diffusion coefficient of macromolecules could be obtained from the spectrum of scattered light. However, the lack of spatial coherence and the non-monochromatic nature of conventional light sources rendered such experiments impossible until 1964 when H. Cummins, N. Knable and Y. Yeh used an optical-mixing technique to resolve spectrally the light scattered from dilute suspensions of polystyrene latex spheres.