Physical Biology

The following sections are included:

• Contents

• Prologue

The life sciences are in transition, with the beginning of the 21st century symbolically marking the initiation of this redefinition. It is a creative, exciting repositioning because it derives from a changed perspective on what is possible. The appearance of the human genome sequence at the turn of the millennium ushered in the post-genomic era, an era of new possibilities, new challenges and new paradigms…

The following sections are included:

• Acknowledgments

• References

The integration of physics and chemistry with biology — physical biology — offers new opportunities for the deciphering of its complexity. Because of the collective interactions of the many elements involved, 4D visualization of structural dynamics is essential to an understanding of the mechanism of the function. Here, we provide an overview of the principles of 4D visualization and highlight the potential of space–time imaging through some examples of applications, ranging from chemical reactions and phase transitions to molecular assemblies and biological cells. Some "big questions" are raised in the hope that the new tools and concepts will provide an understanding of what complexity, and emergence, actually mean.

Of the three kinds of primary beams (neutrons, X-rays and electrons) suitable for structural imaging, the most powerful are coherent electrons. The source brightness of such electrons significantly exceeds achievable values for neutrons and X-rays: their minimum probe diameter is as small as 0.1 nm, and their elastic mean-free path is ca 10nm (for carbon), much less than for neutrons and X-rays. Moreover, because electrons are focusable and may be pulsed at sub-picosecond rates, and have appreciable inelastic cross-sections, electron microscopy (EM) yields information in four distinct ways: in real space, in reciprocal space, in energy space and in the time domain. Thus, besides structural imaging, energy landscapes of macromolecules may be explored, and under optimal conditions elemental compositions, valence states and 3D information (from tomography) may be retrieved by time-resolved EM.

Advances in designs of aberration-corrected high-resolution electron microscopes have greatly enhanced the quality of structural information pertaining to nanoparticle metals, binary semiconductors, ceramics and complex oxides. Moreover, electron tomography sheds light on the shape, size and composition of bimetallic catalysts attached to nanoporous supports. With energy-filtered electron tomorgraphy, chemical compositions of sub-attogram quantities located at the interior of microscopic objects may be retrieved non-destructively.

Radiation damage is the main problem which prevents the determination of the structure of single biological macromolecules at atomic resolution using any kind of microscopy, irrespective of the primary beam. Great advances have recently been made via the EM of biological macromolecules and machines embedded in vitreous ice. Electron cryomicroscopy is now used to analyze the structures of molecules arranged as crystals or as single particles; and cryoelectron tomography reveals nuclear pore complex structure and dynamics.

Electron energy loss spectroscopy and imaging, especially energy-filtered (S)TEM tomography is an important development in the imaging of both inorganic and biological systems; and Zewail's revolutionary single-electron (4D) ultrafast-EM and diffraction contribute richly to the structural imaging of both atomic and macromolecular species at sub-picosecond time scales.

Sixty years after the beginning of the molecular biology revolution, physical scientists are becoming attracted to biology again. This time, unlike the situation six decades ago, we know a great deal about the parts and components of the cell. Moreover, the complexity that drove away many physical scientists in the past is now yielding to the reductionist approach, and complexity in itself has become a central research theme in physics. The stage is thus set for the development of renewed efforts at quantification in biological research and at modeling integrated cellular functions. This is the new playground where physical biology and their practitioners will make their impact. Some of the research challenges lying ahead are discussed.

Most biological macromolecules function in the form of molecular assemblies. Although often conformationally heterogeneous, these assemblies contain rigid subcomplexes whose structures may be determined by cryoelectron microscopy and image processing. We have proposed the use of heavy atom clusters to facilitate the analysis, and have recently synthesized clusters of the size and reactivity towards proteins required for the purpose.

In considering the future of X-ray analysis in 1945, J.D. Bernal predicted that "pure" X-ray analysis would solve more and more complex structures, while "applied" X-ray analysis would become indispensable in a diverse range of fields. This vision has been largely realized, particularly in biology, where X-ray crystallography has contributed substantially to our present understanding of macromolecular structure and function. What remains for the next 50 years? It seems inevitable that advances in X-ray analysis, propelled by developments in biological methodologies, X-ray technology, computation and the tools of physical biology, will solve larger and more complex biological structures, at ever higher resolutions and on faster time scales. A long-term goal will be to use these insights to develop molecularly explicit models with predictive capabilities for the structures and dynamics of cells and higher order assemblages, corresponding to the biological counterpart of weather forecasting.

The genetic code, elucidated in the 1960s through the work of Nirenberg, Ochoa, Khorana and their coworkers, provides a set of molecular instructions for translating nucleic acids into proteins. Codons are assigned to amino acids through high-fidelity charging of transfer RNAs, and through accurate base-pairing between charged tRNAs and messenger RNA. Over the last decade, cells have been outfitted with modified translational machinery that enables the participation of an expanded set of amino acids in protein synthesis. These developments have stimulated a unified view of the chemistry of natural and synthetic macromolecules, and provided a basis for powerful new approaches to materials design, protein evolution, biological imaging, and proteome-wide analysis of cellular processes.

The following sections are included:

• Introduction

• What are the Difficulties?
  • "Swatting Mosquitoes"
  • "If You can't Measure It, You can't Manage It"
  • Theory, Computation, and Simulation

• What is New? Back to the Future with Isothermal Titration Calorimetry (ITC)

• A Model System: Carbonic Anhydrase and Arylsulfonamides
  • Binding of p-R-C₆H₄SO₂NH₂ to Carbonic Anhydrase
  • Entropy-Enthalpy Compensation

• Assessment

• What Do We Need?

• Acknowledgments

• References

Science was once called "natural philosophy" and had as its purview the scientific study of all of nature. Increasing specialization led to a splintering of natural philosophy into a number of separate disciplines and one of the outcomes of this trend was that physics emerged largely as the study of inanimate matter. This peculiar state of affairs imposed an unnatural barrier which largely prevented physicists from seeing the study of living matter as part of their core charter. Similarly, the style of analysis favored in the life sciences was often descriptive, isolating much of the biological mainstream from quantitative descriptions as the rule rather than the exception. An exciting outcome of the biological revolution of the last 50 years is that the study of living matter is emerging as a true interdisciplinary science that will enrich traditional physics and biology alike. I examine some of the philosophical underpinnings of physical biology and then illuminate these ideas through several case studies that highlight the interplay between quantitative data and the models set forth to greet them. One of the interesting outcomes of an analysis from the physical biology perspective is that topics that seem very distant biologically are next door neighbors in physical biology.

Simulating conformational changes in proteins is of great importance and molecular dynamics simulations are expected to play a major role. However, such simulations are hampered by the very short time they can run. Here we present a complex strategy for alleviating this problem. We characterize the structure of proteins by a number of collective variables or order parameters and calculate the associated free energy surface. In particular, we present two efficient methods for exploring this surface, one being well-tempered metadynamics and the other a path-based method. With the help of these methods, very long time scale phenomena can be simulated.

Proteins are highly dynamical entities. The folding, function and higher order assembly of biomacromolecules are key elements of the organization of living matter. Energy landscape theory is illuminating many of these issues of biomolecular dynamics. Folding is dominated by a funnel-like energy landscape with minimal ruggedness. Quantifying this notion using statistical mechanics has led to algorithms for learning about protein energetics using bioinformatics, designing novel proteins and predicting protein tertiary structure from sequence. The funneling of the landscape allows robust predictions to be made for folding kinetics and mechanism. The functional landscape is intimately connected with folding and residual frustration. Binding sites are often frustrated and can be located using algorithms based on energy landscape theory. The higher order assembly of cells is, however, far from equilibrium and requires new concepts beyond current energy landscape theories.

The manner in which a newly synthesized chain of amino acids folds into the unique structure of a functional globular protein depends both on the intrinsic properties of the amino acid sequence and on multiple influences within the crowded cellular milieu. Moreover, if proteins misfold, or fail to remain correctly folded, a common consequence is aggregation and the formation of amyloid deposits, a phenomenon that is implicated in many highly debilitating and increasingly common medical disorders, including Alzheimer's disease and Type II diabetes. In this chapter, we describe how the concerted application of a wide range of the experimental and theoretical techniques of physics and chemistry under laboratory conditions has allowed the fundamental principles of protein folding, misfolding and aggregation to be understood at an atomic level. We then discuss how recent approaches, initially using cells and then model organisms such as the fruit fly, enable the links between these physical and chemical principles and the molecular events that occur in a living system to be explored in a quantitative manner. The results provide compelling evidence for a common underlying origin of the amyloid-associated diseases based on an inherent tendency of functional proteins to convert into the generic alternative and frequently pathogenic amyloid form of structure. They also suggest strategies for the prevention and treatment of these diseases, and provide insight into the way in which the need to fold correctly and to avoid aggregation under physiological conditions has influenced the evolution of protein sequences and the properties that they encode.

A new approach to biology has emerged over the past ten years or so termed systems biology. This approach will not only transform how we approach biology, but how we understand and deal with disease as well. I will discuss why systems biology will transform biology in the 21st century, the emergence and essential principles of systems biology and then focus on its impact on medicine and how it will transform our current largely reactive medicine to a medicine that is predictive, personalized, preventive and participatory (P4). I will discuss this in a somewhat personal manner since I have been so intimately involved with the conceptualization and proof of principle for systems biology for the past 20 some years and for P4 medicine for the past five years.

In recent years, the mystery of consciousness and its material basis has attracted scientific interest beyond the traditional field of philosophy. The neurobiological approach to consciousness aims at identifying its physico-chemical basis at the neuronal level of the brain. Electrophysiological, psychophysical, and functional imaging studies in humans and non-human animals have allowed brain scientists to narrow down on the neural substrates of consciousness and conscious perception. These findings, complemented by the development of a robust theoretical predictive framework, could eventually lead to a rational understanding of the phenomenon of consciousness.

Advances in chemical theory, structural biology, and computer technology continue to increase the power of physics-based methods for biomolecular modeling. Computer simulations at the molecular level have recently played a key role in the discovery of the first in a new class of antiviral drugs for HIV/AIDS. Simulations at the supramolecular and cellular level will play an increasing role in drug discovery in the coming years.

Is biology a quantitative science like physics? In this chapter I will discuss the role of precision measurement in both physics and biology, and argue that in fact both fields can be tied together by the use and consequences of precision measurement.

The following sections are included:

• Introduction

• My Early Studies: The K⁺ Channel Signature Sequence

• The KcsA Structure and Selective K⁺ Conduction

• Common Structural Principles Underlie K⁺ and Cl^- Selectivity

• Trying to See a K⁺ Channel Open and Close

• Concluding Remarks

• Acknowledgments

• References

Chemistry abounds with examples of molecules that have broken symmetry, such as ammonia, which is not flat but oscillates between two identical "umbrella" conformations. Benzene in its ground state is a flat symmetrical ring, but has lowered symmetry in its first excited state. What is in evidence here is competition between energy of electronic coupling between nuclei, tending to delocalize, and energy of electronic-vibrational coupling, tending to localize. In the fully delocalized symmetrical state we have a static charge distribution, whereas in the strongly localized case there is dynamic equilibrium between equivalent structures, with a measurable thermal exchange rate constant. Electron transfer processes, including critical biological ones, are governed by these restraints. The history is intriguing.

It has been known since the 1970s how one can in principle use classical molecular dynamics (i.e., numerically computed classical trajectories) as input to a semiclassical (SC) theory that provides a good description of all quantum mechanical effects in the dynamics of molecular systems. Over the last decade, various initial value representations of SC theory have been shown to provide a practical way for implementing these SC approaches for large molecular systems, those of interest for applications in bio-molecular and molecular-materials areas.

The following sections are included:

• Biographies