MARCO TEÓRICO, HIPÓTESIS Y OBJETIVOS DE LA INVESTIGACIÓN
C) Dentina terciana:
2.1.6 FORMOCRESOL 1 Definición.
The similarity between the purple non-sulphur bacteria and PS II has been suggested many times following a variety of observations. Both
reaction centres have a similar complement of cofactors including a pair of pheophytins (Tiede et al. 1976); similar electron transfer rates (see section
1 .8 .6 ); the ability to bind herbicides, in particular the photoaffinity
labelling by azidoatrazine (Pfister et al. 1981). Spectroscopically the two systems show similarities both having a Q^-Fe-Qg acceptor side th at is magnetically coupled.
The amino acid sequences of the D l and D2 subunits are available in a large number of organisms (reviewed by Svensson et al. 1991) and there is significant homology between the sequences and between them and those from the purple bacteria.
There have been many studies and several reviews on this analogy and its evolutionary implications (Deisenhofer and Michel, 1989b;
Beanland, 1990 and Nitschke and Rutherford, 1991).
As discussed in sections 1.8.6 and 1 .8 . 8 the photochemical differences
in function between the two types of reaction centre are in the donor potential and on the lumenal water splitting activity of the complex.
Physiological differences include the sensitivity of PS II to bicarbonate depletion (see section 1.8.8) and photoinhibition (see section 1.8.9).
1.10 Aims of this thesis
This work sets out to investigate,
1) the structure and function of the PS II reaction centre by the
construction of a computer generated model based on the Q-type reaction centre structures from purple non-sulphur bacteria.
2 ) the evolutionary relationships between different photosynthetic
organisms and the ancestral relationship between the subunits in the reaction centre.
3) the binding of the bicarbonate cofactor to the stromal side of PS II. 4) a possible model structure of the water oxidizing complex and a binding site for such a manganese tetram er on the lumenal side of the PS II model.
5) the interaction of the herbicide DCMU with the modelled Qg binding site.
6 ) the protein-cofactor interactions in the heterodimer and the possible
effects on the route of electron transfer.
7) the use of EPR to determine the structure-activity relationship of known and experimental herbicides.
8 ) the orientation of the cytochrome 6 5 5g monomers and the tertiary and
quaternary structure of cytochrome multimers by analysis of sequence and secondary structure information as well as building three-dimensional models of the transmembrane spans.
9) the psbl gene product; with respect to its sequence, secondary
structure analysis and generation of a three-dimensional model structure of the predicted a-helix.
10) the effect of aerobic, anoxic and anaerobic conditions on the loss of PS II activity due to pbotoinbibition, in preparations with and vsdtbout native bicarbonate.
11) the g=3 EPR signal from cytochrome bggg in various PS II preparations.
2 M aterials and M ethods
2.1 Knowledge based m odelling
The need for detailed information of protein structures has
highlighted the discrepancy between the numbers of primary sequences and the number of tertiary structures th at are solved. The Brookhaven database (Bernstein et al. 1977) has over 20,000 partial or whole sequences of DNA or proteins but there are only about 500 three-dimensional protein structures solved to high resolution. The availability of computer data storage and retrieval systems and high capacity processing has made computer modelling of three-dimensional structures possible during the last decade.
It was elegantly demonstrated by Anfinsen et al. (1961) th a t some enzymes hold in their primary sequence all of the information to fold into their tertiary structure. Techniques have been developed to model protein structures directly from their sequences ah initio but the success of these techniques is limited as the protein folding process seems to be very
complex (reviewed by Fasman, 1989 and Thornton et al. 1991). Membrane proteins as a subset of all proteins have a series of characteristics th a t have been thoroughly reviewed (Jennings, 1989 and Singer, 1990).
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1 . 1Homology m odelling
Homology modelling relieson the fact th at polypeptides within a family group of proteins have similar function, similar sequences and
[described as a series of discrete methodologies (described in Blundell et al.
1987 and Lee, 1992).
The sequence to be modelled is used to search databases to find all of the related sequences and structures th at are available. The sequences are then aligned. There are many tried and tested alignment programs based on very similar methods th at generally give similar results providing there is enough similarity between the sequences (reviewed by Feng et al.
1985).
The critical step in the modelling process is the alignment of the sequence to be modelled to the sequence or sequences of the structure or structures available. Traditionally this has been achieved purely using the sequence alignment.
Once aligned to the structures, sections of the sequence to be modelled were chosen to be structurally conserved regions (SCRs). The conformation of the model protein was then set to be the same as one of the reference proteins. The conformation of the side-chains is either set as in the reference structure, set by an independent regime of conformation rules or set by hand and energy minimized.
The regions between the SCRs are known as structurally variable regions (SVRs), these are those part of the polypeptide th a t do not bear strong homology to the reference proteins. These loops are built either by database searches (discussed in Jones and Thirup, 1986) or by loop
generation methods (Shenkin et al. 1987). The SVRs are fitted to the SCRs generally by a least squares fitting procedure.
The terminal regions may be added before the final energy
minimization. The process of energy minimization moves parts of the protein to relieve strain imposed by side-cbain clashes and bad peptide chain topologies. The overall energy of the structure can be further reduced by repositioning side-chains to form bridges and hydrogen bonds.
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