Biomolecular structure investigations

Within five years of the verification of VCD in solutions, first attempts were made to apply the technique to study biomolecular solution conformation in aqueous solution. The first samples studied as solutions in D.O were simple amino acids and di-and tripeptides [26]. However, due to instrumental limitations, VCD was collected only in the C-H and N-H stretching region (2.5 to 3.5 fim). Although these early studies were necessary for the development of the experimental technique, it was not until experimental advances allowed the detection of VCD in the 6 fxm region that the full potential of VCD was realized for peptide structural investigations. 

Protein crystal structures are archived in the Protein Data Bank (PDB) (Bernstein et al. 1977 Berman et al. 2000). About 5 per cent of the approximately 14 000 (December 2000) entries ( 12 500 proteins, peptides, and viruses, 900 nucleic acids, 600 pro-tein/nucleic acid complexes, 20 carbohydrates) contain the qualifier form in the compound name/descriptor field, and most of those refer to polymorphic varieties. In biomolecular crystallography great efforts are expended varying crystallization conditions in the attempts to obtain single crystals suitable for structural investigations... 

It should also be noted that the method of surface attachment may also have an influence the properties of the biomolecule of interest. For example, while covalent attachment may facilitate the structural investigation of isolated biomolecular species, such a strong attachment may be disadvantageous if the user wishes to subsequently visualize any dynamic processes of this molecule, as they may be hindered/prevented. For the observation of such dynamic processes the use of immobilization methods such as weak electrostatic interaction is therefore more usual, and in this type of experiment thorough investigation and optimization of the immobilization process is absolutely essential. 

Biological membranes provide the essential barrier between cells and the organelles of which cells are composed. Cellular membranes are complicated extensive biomolecular sheetlike structures, mostly fonned by lipid molecules held together by cooperative nonco-valent interactions. A membrane is not a static structure, but rather a complex dynamical two-dimensional liquid crystalline fluid mosaic of oriented proteins and lipids. A number of experimental approaches can be used to investigate and characterize biological membranes. However, the complexity of membranes is such that experimental data remain very difficult to interpret at the microscopic level. In recent years, computational studies of membranes based on detailed atomic models, as summarized in Chapter 21, have greatly increased the ability to interpret experimental data, yielding a much-improved picture of the structure and dynamics of lipid bilayers and the relationship of those properties to membrane function [21]. 

Third, as the size and complexity of the biomolecular systems at hand further expand, there are more uncertainties in the molecular model itself. For example, the resolution of the X-ray structure may not be sufficiently high for identifying the locations of critical water molecules, ions and other components in the system the oxidation states and/or titration states of key reactive groups might be unclear. In those cases, it is important to couple QM/MM to other molecular simulation techniques to establish and to validate the microscopic models before elaborate calculations on the reactive mechanisms are investigated. In this context, pKa and various spectroscopic calculations [113,114] can be very relevant. 

Lyotropic lamellar (La) liquid crystals (LC), in a form of vesicle or planar membrane, are important for membrane research to elucidate both functional and structural aspects of membrane proteins. Membrane proteins so far investigated are receptors, substrate carriers, energy-transducting proteins, channels, and ion-motivated ATPases [1-11], The L liquid crystals have also been proved useful in the two-dimensional crystallization of membrane proteins[12, 13], in the fabrication of protein micro-arrays[14], and biomolecular devices[15]. Usefulness of an inverted cubic LC in the three-dimensional crystallization of membrane proteins has also been recognized[16]. 

Proteins interact with each other in many different ways. These interactions may be structural, evolutionary, functional, sequence based, and metabolical. Life depends on such biomolecular interactions. Among all these interactions, structural ones are the simplest and easiest to investigate because they are the most definite. Therefore, the main goals of bioinformatics are to create and to maintain the databases of the biological information which may lead to better understanding of these interactions. 

Fig 9.29 illustrates Zn determination in a Zn binding protein in a selected protein spot after separation of the proteins of an S layer used as biomolecular template, as described above, by 2D gel measured by LA-ICP-SFMS at medium mass resolution (m/Am = 4000). ° The Zn ion intensity in the protein spot (compared with the background signal in the gel blank) was measured at three different positions in the selected spot. Using LA-ICP-SFMS, the highest Zn intensity was found in the middle of the protein spot investigated. Additional measurements by MALDI-MS on Zn containing protein spot in 2D gel can be useful to identify the structure of the protein. ... 

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