Biochemical experimental approaches

For molecules of molecular weight above 20,000 g/mol, X-ray diffraction remains the only experimental approach available to obtain detailed and reliable three-dimensional atomic models. The major steps of the method include the obtention of large and well-ordered crystals, their exposure to X-rays and collection of diffraction data and the phasing of these data to obtain by Fourier analysis a three-dimensional view (or map) of the electron density of the molecule. Finally a three-dimensional atomic model of the protein is fitted like a hand in a glove within this map, using a kit containing all the available biochemical and spectroscopic information (Table 6.2). The reliability of the final atomic model is of course dependent on the qnality of the electron density map. This qnality depends on the number of X-ray data per atom and on the resolution and accnracy of these data, which in turn are highly dependent on the size and quality of the crystals. 

In fact, it is probably fair to say that very few problems involving real momentum, heat, and mass flow can be solved by mathematical analysis alone. The solution to many practical problems is achieved using a combination of theoretical analysis and experimental data. Thus engineers working on chemical and biochemical engineering problems should be familiar with the experimental approach to these problems. They have to interpret and make use of the data obtained from others and have to be able to plan and execute the strictly necessary experiments in their ovm laboratories. In this chapter, we show some techniques and ideas which are important in the planning and execution of chemical and biochemical experimental research. The basic considerations of dimensional analysis and similitude theory are also used in order to help the engineer to understand and correlate the data that have been obtained by other researchers. 

Despite its limited size, this book has a fairly broad scope it starts with access to the literature of biochemistry, sets out the main features of the organisation of a biochemical laboratory, discusses a range of laboratory methods, both general and more specialised and concludes with coverage of some advanced instrumental techniques. It is not a practical handbook in the sense that experiments are described, or experimental protocols are presented in detail, rather it contains the information necessary for the reader to plan experiments and to be in a position to appreciate what conclusions could in principle be drawn from particular experimental approaches. It is also one of our aims to stimulate the reader to use methods that they may not be currently very familiar with. For reasons of space, we have been able to describe methods in detail in only a limited number of cases, and for this we have selected core techniques, which find wide application. Elsewhere, we have had to rely on the literature to take matters further in doing this, we have given preference to widely accessible journals, reviews and more recent monographs. 

Naturally, the hypothetical model presented possesses its own peculiar problems in terms of experimental approaches, and any definitive confirmation or disconfirmation of the model would seem to depend on the development of very much more sophisticated biochemical and ESR techniques. We felt, however, that at least parts of the theory presented could be subjected to an operational test. The experimental procedures involved in this test, and the at least partially affirmative results obtained, are the subject matter of the next chapter. 

Still, in a system with potentially huge degrees of freedom such as life, the construction in a computer may miss some essential factors. Hence, we need the third experimental approach—that is, construction in a laboratory. In this case again, one constructs a possible biology world in a laboratory, by combining several procedures. For example, this experimental constructive biology has been pursued by Yomo and co-workers (see, e.g., Refs. 18-21 at the levels of biochemical reaction, cell, and ensembles of cells). 

Of course, the interaction between the membrane-spanning helices A and B that is presumably stabilized by these carotenoids may in turn influence the trimer formation of the complex. However, it is also possible that the additional carotenoid(s), known to be part of the complex from biochemical data but not visible in the crystal stmcture, stabilize trimers. This can be suggested from the observation that in the aba mutant of Arabidopsis where zeaxanthin appears to replace violaxanthin and neoxanthin, the major LHCII dissociates more easily into monomers when isolated under partially denaturing conditions (Tardy and Havaux, 1996). A closer biochemical inspection of the major LHCII in various carotenoid-deficient plant and algae mutants will be necessary to assess the impact of carotenoids on the formation of trimeric LHCII. Another experimental approach will be to study how the variation of the carotenoid components influences the reconstitution of trimeric LHCII in vitro (Hobe et al, 1994). 

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