Structure Determination of Biopolymers

There are four levels of structure observed in proteins [9] and it is important that once the molecular weight of a protein has been determined that these are also elucidated in order that the way in which the protein functions may be understood. 

MS may be used to investigate all of these but by far the most common application of LC-MS is concerned with the determination of amino acid sequences. 

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The structure determination of biopolymers using NMR spectroscopy usually involves interactions of protons[25,1221. Typically, interactions of protons (nuclear Over-... 

The structure determination of biopolymers using NMR spectroscopy usually involves interactions of protons[216,33. Typically, interactions of protons (nuclear Overhauser effect, NOE) that are close in space but separated by several subunits of the biopolymer are used to establish the folding of the backbone. Distance restraints are then used to compute a structure which is checked by back-calculation of the NOE spectra and comparison with experimental results 361. For large and highly flexible systems molecular dynamics is invaluable for scanning the conformational space. 

Quadrupole ion traps ions are dynamically stored in a three-dimensional quadrupole ion storage device (Fig. 10.6) [37]. The RF and DC potentials can be scanned to eject successive mass-to-charge ratios from the trap into the detector (mass-selective ejection). Ions are formed within the ion trap or injected into an ion trap from an external source. The ions are dynamically trapped by the applied RF potentials (a common trap design also makes use of a bath gas to help contain the ions in the trap). The trapped ions can be manipulated by RF events to perform ion ejection, ion excitation, and mass-selective ejection. This provides MS/MS and MS experiments, which are eminently suited for structure determinations of biopolymers [38] (see Section 10.4). 

For structural determination of biopolymers, multidimensional (three or more) NMR spectroscopy is helpful, as the spectrum carries a number of... 

A relatively common feature of many problems involving molecular weight determination of biopolymers is that of association-dissociation equilibrium. Subunit structure of enzyme proteins is well recognized (1), and methods of dissociation of subunits to obtain monomer molecular weight are widely utilized (2). A previous paper described the application of an equilibrium gel partition method to the analysis of macromolecular association in a monomer-dimer case (3). The experimental parameters in a system utilizing the Sephadex series of gel filtra-... 

This collection of papers was part of a unique symposium held during the 178th Meeting of the American Chemical Society. The symposium, Diffraction Methods for Structural Determination of Fibrous Polymers, had a pronounced international character, with scientists from 12 different countries. The speakers represented both the synthetic polymer and biopolymer fields, with contributions in each of the three classes of natural polymers nucleic acids, proteins, and polysaccharides. Most important, the symposium centered on methods and techniques for studying fibrous polymers, methods that are usually taken for granted despite their inadequacies. 

R595 H. Zhou, A. Vermeulen, F. M. Jucker and A. Pardi, Incorporating Residual Dipolar Couplings into the NMR Solution Structure Determination of Nucleic Acids , Biopolymers, 1999-2000, 52, 168... 

The biological function of biopolymers such as polypeptides, proteins, nucleic acids etc. depends strongly on their ordered structure which is determined by the pattern of inter- and intramolecular interactions given by the primary structure. 

The application areas for LC-MS, as will be illustrated later, are diverse, encompassing both qualitative and quantitative determinations of both high-and low-molecular-weight materials, including synthetic polymers, biopolymers, environmental pollutants, pharmaceutical compounds (drugs and their metabolites) and natural products. In essence, it is used for any compounds which are found in complex matrices for which HPLC is the separation method of choice and where the mass spectrometer provides the necessary selectivity and sensitivity to provide quantitative information and/or it provides structural information that cannot be obtained by using other detectors. 

Determination of protein secondary structure has long been a major application of optical spectroscopic studies of biopolymers (Fasman, 1996 Havel, 1996 Mantsch and Chapman, 1996). These efforts have primarily sought to determine the average fractional amount of overall secondary structure, typically represented as helix and sheet contributions, which comprise the extended, coherent structural elements in well-structured proteins. In some cases further interpretations in terms of turns and specific helix and sheet segment types have developed. Only more limited applications of optical spectra to determination of tertiary structure have appeared, and these normally have used fluorescence or near-UV electronic circular dichroism (ECD) of aromatic residues to sense a change in the fold (Haas, 1995 Woody and Dunker, 1996). 

Nowadays it is well established that the interactions between different macromolecular ingredients (i.e., protein + protein, polysaccharide + polysaccharide, and protein + polysaccharide) are of great importance in determining the texture and shelf-life of multicomponent food colloids. These interactions affect the structure-forming properties of biopolymers in the bulk and at interfaces thermodynamic activity, self-assembly, sin-face loading, thermodynamic compatibility/incompatibility, phase separation, complexation and rheological behaviour. Therefore, one may infer that a knowledge of the key physico-chemical features of such biopolymer-biopolymer interactions, and their impact on stability properties of food colloids, is essential in order to be able to understand and predict the functional properties of mixed biopolymers in product formulations. 

The term food colloids can be applied to all edible multi-phase systems such as foams, gels, dispersions and emulsions. Therefore, most manufactured foodstuffs can be classified as food colloids, and some natural ones also (notably milk). One of the key features of such systems is that they require the addition of a combination of surface-active molecules and thickeners for control of their texture and shelf-life. To achieve the requirements of consumers and food technologists, various combinations of proteins and polysaccharides are routinely used. The structures formed by these biopolymers in the bulk aqueous phase and at the surface of droplets and bubbles determine the long-term stability and rheological properties of food colloids. These structures are determined by the nature of the various kinds of biopolymer-biopolymer interactions, as well as by the interactions of the biopolymers with other food ingredients such as low-molecular-weight surfactants (emulsifiers). 

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