Introducing the Time-resolved methods in biophysics series

The introduction of modern physico-chemical methods in the study of biologically relevant processes has boosted a dramatic progress in the quantitative description of the mechanistics of fundamental biological events. Biological processes are dynamic in nature. For instance, fluctuations in biomolecular structures are essential to function and propagate one equilibrium structure to the next, passing through intermediate structures that may be metastable and short-lived. Thus, time resolution is important to biomechanistic studies, because biomolecules are dynamic structures.

Biology, as other fields, benefited to a great extent from some of the major research topics of the last century, i.e. radioactivity, space research, nuclear physics, and high-energy physics. For decades, these research fields have developed the ideas and a large part of the instrumentation to observe events with an ever rising time-resolution—up to the very extreme of detecting processes much too fast to be detected by their product signature only.

From a technical point of view, the field is rapidly changing with new time-resolved experimental methods being frequently introduced. Sometimes even old methods gain new momentum by the combination with modern tools molecular biologists have developed.

State of the art structural methods can give access to atomic resolution. Although crystallographic and spin resonance techniques can provide more structural detail, optical methods offer important advantages in versatility, specificity, and time resolution.

There are three cases, where optical spectroscopy is extremely helpful in the study of biological samples. 1. When investigating systems that are far from being studied successfully by (steady-state) structure-determining methods. 2. When optical spectroscopy—especially its superior time resolution—adds information about a biological specimen even if the static structural information has been obtained. 3. When time-resolved structural information can be obtained but the very unnatural environment (e.g. the high concentrations in solution (NMR) and high concentration together with ordered arrangement in crystals (X-ray diffraction)) affects the biophysical behaviour of the investigated system.

There are only a few Nobel prizes awarded directly for the introduction and the use of an optical spectroscopy (like the one G. Porter shared with R. G. W. Norrish and M. Eigen, “for their studies of extremely fast chemical reactions, effected by disturbing the equilibrium by means of very short pulses of energy”†). One of the most prominent subjects to be studied by optical spectroscopy—although not awarded by a Nobel Prize directly—is photosynthesis. Photosystems are complex biological devices that are able to convert visible light into an electrochemical potential. The time course, energetics as well as the molecular connections have been solved years before the (static) biomolecular structure(s) have been enlightened. In addition, the time-resolved methods envision the limits of a static structure to explain reactions of dynamic systems.

The information from time-resolved spectroscopic methods has sometimes provided the key to introduce new concepts and open real breakthroughs in the way we understand macromolecular function. A typical example is ligand binding to myoglobin. Already in the 70s, the seminal work by Frauenfelder and coworkers introduced the concepts of energy landscape and conformational substates to interpret the experimental observation of non-exponential ligand rebinding following photolysis of myoglobin–CO complexes at temperatures below 200 K.¹ The inverse temperature dependence of the rebinding kinetics observed at higher temperatures was rationalized much later when more refined structural informations became available,² showing that after photolysis CO could be docked into the Xe cavities.³ From then on, the interplay between time-resolved spectroscopic^4–7 and structural^8–10 investigations has lead us to a substantial advancement of our understanding of the mechanistics of ligand binding. This interplay between structural and spectroscopic information shows that either approach is not in itself sufficient to provide a complete view on the details of the mechanism by which the ligand is bound by the haem Fe. It is a wonderful example of how very different experimental methods converge to provide a comprehensive view of the phenomena.

Another example is the elucidation of the primary processes in the most important biological photoreceptors of animals (rhodopsin) and plants (phytochrome). It nicely demonstrates, how the development of new time-resolved optical spectroscopy methods refines the exactness and completeness of the knowledge about the—in this case ultrafast (<10 ps)—underlying molecular processes. By the combined results of ultrafast time-resolved visible absorption, fluorescence, infrared absorption and resonance Raman spectroscopy detailed pictures for the reaction mechanism—involving a trans–cis isomerization of the chromophore—were established for the different photoreceptors.^11–17 This information is very useful despite the emerging structural data.

The series of papers on time-resolved methods in biophysics aims at giving state of the art descriptions of time-resolved experimental approaches to the characterization of relevant biophysical and biochemical processes. The first papers in the series are derived from the lectures delivered at the X School of Pure and Applied Biophysics, “Time-resolved spectroscopic methods in biophysics”, held in Venice at Palazzo Franchetti, in January 2006. This school was organized by Thomas Gensch and Cristiano Viappiani on behalf of the Società Italiana di Biofisica Pura ed Applicata (SIBPA) and the Istituto Veneto di Scienze, Lettere ed Arti (IVSLA). Information on the school is available at http://sibpa.itc.it/pages/scuola_2006.html.

The contents of the school covered consolidated time-resolved spectroscopy methods as well as frontier techniques. Time-resolved absorbance methods in the visible and in the infrared spectral range, with time resolution extending from a few femtoseconds out to many milliseconds, were dealt with the emphasis on the biological applications. Time-resolved vibrational spectroscopies touched important techniques as time-resolved FTIR, 2D femtosecond IR, and resonance Raman. Luminescence methods included near IR phosphorescence from singlet oxygen, single molecule fluorescence spectroscopies, fluorescence lifetime imaging and fluorescence correlation spectroscopy.

The first two papers in the series appear in this issue of Photochemical & Photobiological Sciences. The first paper, by Naumann et al.,¹⁸ reports a pump and probe surface-enhanced resonance Raman approach for studying biological photoreceptors. The paper by Abbruzzetti et al.¹⁹ deals with the applications of nanosecond flash photolysis with time-resolved absorbance detection to the study of ligand binding to haem proteins.

While the suggestion for this collection of papers emerged from the lectures delivered at the Venice school, this series intends to extend beyond the contents of the school, and will hopefully host more and more papers in the future, dealing with time-resolved approaches, not necessarily restricted to spectroscopy. It is our hope that papers in this series will become a useful reference for researchers tackling, with time-resolved methods, the sometimes difficult problems posed by chemistry and biology.

Thomas Gensch

Institut für Biologische Informationsverarbeitung 1

Forschungszentrum Jülich, Jülich,Germany

Cristiano Viappiani

Dipartimento di Fisica

Università degli Studi di Parma

Parma, Italy

References

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2. A. Ostermann, R. Washipky, F. G. Parak and G. U. Nienhaus, Ligand binding and conformational motions in myoglobin, Nature, 2000, 404, 205–208.
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19. S. Abbruzzetti, S. Bruno, S. Faggiano, E. Grandi, A. Mozzarelli and C. Viappiani, Time-resolved methods in Biophysics. 2. Monitoring haem proteins at work with nanosecond laser flash photolysis, Photochem. Photobiol. Sci., 2006, 5 10.1039/b610236k.

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Footnote

† From the announcement of the Nobel prize (http://nobelprize.org/nobel_prizes/chemistry/laureates/1967/). Although the motivation for the work that lead to the Nobel prize was common, Norrish and Porter introduced flash-photolysis to study fast photochemical reactions, while Eigen applied electrical pulses to induce fast heating of solutions and monitor the subsequent relaxation of the perturbed chemical equilibria.

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