BIOMACROMOLECULAR STRUCTURE PROTEINS

CHAPTERS BIOMACROMOLECULAR STRUCTURE PROTEINS TABLE 5.8 continued... 

Several factors must be considered for a particular biomacromolecular structure application that will affect the choice of spectroscopic methods. These include structural resolution necessary, chemical nature of biomacromolecule (protein, nucleic add, or glycan), amount/concentration of biopolymer available, sample preparation (solid or solution), solvents of interest, and desired structure information (secondary or tertiary structure). Structural resolution varies considerably for the various spectroscopic methods, with X-ray diffraction and NMR providing atomic resolution (high resolution) and ultraviolet (UV) absorption revealing merely information about the polarity of the chromophore s environment (low resolution). X-ray studies require crystals while NMR experiments prefer solutions in deuterated solvent. Solvent preferences can affect the choice of spectroscopic method as, for example, infrared (IR) encoimters strong interference from water, while optical rotatory dispersion (ORD) and circniar dichroism (CD) do not. Some of the commonly used spectroscopic methods in structural analyses of biomacromolecules will be discussed. 

In proteins, cysteine residues are not always readily accessible, since they are often involved in disulfide bridges within the complex three-dimensional biomacromolecular structures. Therefore, only a small number of cysteine residues can be used for bioconjugation reactions. The ligation of polymer bound dibromomaleimides takes advantage of this circumstance, as it allows for the functionalization of disulfide moieties. Haddleton and coworkers demonstrated the applicability of this reaction for bioconjugation of salmon calcitonin (sCT). ... 

There is considerable interest in the coordination" and catalytic chemistries of these discrete clusters. Because of its high electron count, the hexaaquo ion [Ta6(p-Br)i2(OH2)6] has been used frequently for phase determination of isomorphous protein derivatives by SIR, MIR, SIRAS/MIRAS, and SAD/MAD methods in biomacromolecular crystallography. This use is growing as larger biomacromolecular structures and assemblies (e.g., membrane proteins, ribosomes, proteasomes) are studied. 

Water is an essential part in the biomacromolecular system, which is mainly responsible for the structure and functions of nucleic acids, proteins, and other constituents of cell [136-138]. Both proteins and DNA are generally hydrated. It is well known that the conformation of DNA is sensitive to hydration, and presence of salts and ligand molecules [112, 138]. The nucleic acids have three levels of water structure. About 12 water molecules per nucleotide are involved in the primary hydration shell [107, 112, 137, 138]. The water molecules present in the primary shell are impermeable to cations and do not form ice on freezing. The secondary level is permeable to cations and forms ice on freezing and third level is the completely disordered, so-called bulk water. Several theoretical studies have been carried out on the level of hydration on DNA bases, base pairs, base stacks, and double helical DNA [107, 121, 131, 139]. Both the experimental and molecular simulation studies have clearly brought out the importance of hydration in DNA and RNA structures [140-147]. 

The architecture of protein molecules is complex and can be described according to structural organization as primary structure (amino acid sequence), secondary structure (regular structures such as helical, pleated sheet, and coil stractures), tertiary structure (fold in three-dimensional space), quaternary structure (subunit structure) and quintemary structure (biomacromolecular complexes). Usually the overall three-dimensional (3D) architecture of a protein molecule is termed as its conformation, which refers to its secondary and tertiary structures. Between these two stractures, motifs (supersecondary structures) refer to the packing of adjacent secondary stractures into distinct structural elements and domains refer to identifiable 3D structural units that may correspond to functional units. The structures of most proteins with more than 200 amino acid residues appear to consist of two or more domains. 

The knowledge based structural prediction (Blundell et al, 1987) depends on analogies between a biomacromolecule of known sequence and other biomacromolecules of the same class with known 3D structure at all levels in the hierarchy of biomacromolecular organization. In the numerically based statistical methods, the structural rules and parameters (conformational propensities) for each residue are extracted by statistical analyses of the structural database and used to predict the structure along the sequence of the macromolecule. Examples of the statistical methods that are commonly applied to predict secondary structure and folding preference of proteins will be illustrated. 

When biomacromolecular embedding is considered, such as protein matrices and DNA structures, the discrete formulation is instead to be preferred in those cases a detailed and atomistic description of the macromo-lecular environment is necessary, in order to obtain accurate descriptions of the molecular process of interest. Moreover, for these systems, accurate force fields are generally available. Within this framework, the QM/MM approach is commonly used in combination with MD simulations to both achieve a proper statistical sampling and to account for the effects of fluctuations. Commonly the MD simulations are performed at a fiilly classical level (especially if the systems are large and the time-windows to be explored are long). In the 2010s, however, QM/MM-MD are also becoming feasible for small-medium QM systems for time windows of the order of tens to hundreds ofps. ... 

Experimental dipole moments may be used to check the validity of electrostatic calculations. In the past, dipole moments of proteins were often characterized by measurements of dielectric relaxation. More information may be obtained by measurements of the electric dichroism because these measurements provide not only the magnitude of the dipole moment but also the optical anisotropy with respect to the dipole vector. Thus, measurements of the electric dichroism provide a more rigorous test for calculations of electrostatic parameters of proteins. Using the calculations described earlier for pK s of titratable groups, one can predict dipole moments of proteins and their axes given by the principal axes of the rotational diffusion tensor and compare them with electrooptical data. ° One important aspect of comparison of computed and experimental dipole moment is that computations of dipole moments, optical anisotropy, and rotational diffusion coefficients can be used in combination with experimental electrooptical procedures to determine the long-range structure of biomacromolecular assemblies, such as the complexes of DNA and proteins described by Pbrschke et al. so... 

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