Molecular interactions noncovalent complexes

In liquid chromatography, affinity purification protocols (4-8) have been known for a long time. Naturally, electrophoresis can be used just as well to observe molecular or noncovalent interactions of DNA oligomers, provided the complex has distinct electrophoretic properties different from those of the free molecules. Therefore, affinity capillary electrophoresis (ACE) can be a powerful tool for studying DNA-drug or DNA-biopolymer interactions. Several reviews discussing these aspects of ACE have been published in recent years (9-19). The crucial aspects of DNA in this field are covered comprehensively in a recent overview article (20). 

Pseudomolecular Ions. In contrast to the traditional MS, the highest mass peaks in ESI/APCI spectra are not always the molecular ion of interest. Instead, pseudomolecular ions, or noncovalent complex ions, are commonly observed. The pseudomolecular ions are generally formed by the analyte-adduct interaction in the solution system that is preserved as a result of the soft ionization of the ESI/APCI process. These ions are also formed by analyte-adduct gas-phase collisions in the spray chamber [49]. The exact mechanisms of how the analyte adducts are formed in ESI/APCI still remain unresolved at this point. More often than not, the adduct ion formation is a major cause for the low detection limit for ESEAPCI MS. However, these associative processes have also created interest in the study of drug-protein/ drug-oligonucleotide gas-phase complexes that benefit from the ability of ESI/APCI MS analysis. 

Analogs of biomolecules carrying an attached second component have sometimes been referred to as bioconjugates as well. Furthermore, the term bioconjugation has been used in numerous cases when noncovalent molecular interactions were considered, for example, in the complexation of glycosami-noglycans and proteins such as heparin sulfate and growth factors. 

Protein-protein interactions such as those between antibody and antigen and enzyme and substrate pairs can be studied by MS. Since many cellular functions are performed by protein complexes and not by individual proteins, it is important to be able to identify the interacting protein components. As ESI is a gentle technique, the detection of weakly boimd (noncovalent) complexes in the gas phase is possible. There is evidence to suggest that these ESTMS observations can reflect the expected interactions in solution phase and can even determine the strength of these interactions. Mass spectrometric experiments with multimeric proteins can yield information on both the stoichiometry and molecular nature of subunit interactions. 

Molecular recognition and noncovalent complexes are at the core of reaction networks in biology. Molecular complexes are often associated with the proliferation of disease (see, for example, the Tax-associated complexes in human T-cell leukemia type 1, HTLV-1 [33]). Along with other competing techniques (e.g., surface plasmon resonance), mass spectrometry can be successfully used to detect noncovalent complex formation. The corresponding ions can be present in both MALDl [34] and ESI [35] spectra, although the latter is used more often. A wide variety of protein-protein interactions as well as protein interactions with other species (nucleotides, carbohydrates, etc.) have been studied. The spectra can reveal the components of the complex and in some cases the association constant. 

As a consequence, for accurate molecular mass analysis of intact proteins, ESI in combination with an orthogonal TOE, electrostatic ion trap or FT-ICR MS is the preferred instrumentation if the samples are very clean and not too complex. If this is not the case or sample throughput is more important than mass accuracy, MALDI-TOF MS is the first choice. Another important advantage of ESI is its superior performance for the analysis of noncovalent complexes including DNA-protein interactions. 

Size Isomers. In solution, hGH is a mixture of monomer, dimer, and higher molecular weight oligomers. Furthermore, there are aggregated forms of hGH found in both the pituitary and in the circulation (16,17). The dimeric forms of hGH have been the most carefully studied and there appear to be at least three distinct types of dimer a disulfide dimer connected through interchain disulfide bonds (8) a covalent or irreversible dimer that is detected on sodium dodecylsulfate- (SDS-)polyacrylamide gels (see Electroseparations, Electrophoresis) and is not a disulfide dimer (19,20) and a noncovalent dimer which is easily dissociated into monomeric hGH by treatment with agents that dismpt hydrophobic interactions in proteins (21). In addition, hGH forms a dimeric complex with ( 2). Scatchard analysis has revealed that two ions associate per hGH dimer in a cooperative... 

Finally, to produce the structural and functional devices of the cell, polypeptides are synthesized by ribosomal translation of the mRNA. The supramolecular complex of the E. coli ribosome consists of 52 protein and three RNA molecules. The power of programmed molecular recognition is impressively demonstrated by the fact that aU of the individual 55 ribosomal building blocks spontaneously assemble to form the functional supramolecular complex by means of noncovalent interactions. The ribosome contains two subunits, the 308 subunit, with a molecular weight of about 930 kDa, and the 1590-kDa 50S subunit, forming particles of about 25-nm diameter. The resolution of the well-defined three-dimensional structure of the ribosome and the exact topographical constitution of its components are still under active investigation. Nevertheless, the localization of the multiple enzymatic domains, e.g., the peptidyl transferase, are well known, and thus the fundamental functions of the entire supramolecular machine is understood [24]. 

The final part is devoted to a survey of molecular properties of special interest to the medicinal chemist. The Theory of Atoms in Molecules by R. F.W. Bader et al., presented in Chapter 7, enables the quantitative use of chemical concepts, for example those of the functional group in organic chemistry or molecular similarity in medicinal chemistry, for prediction and understanding of chemical processes. This contribution also discusses possible applications of the theory to QSAR. Another important property that can be derived by use of QC calculations is the molecular electrostatic potential. J.S. Murray and P. Politzer describe the use of this property for description of noncovalent interactions between ligand and receptor, and the design of new compounds with specific features (Chapter 8). In Chapter 9, H.D. and M. Holtje describe the use of QC methods to parameterize force-field parameters, and applications to a pharmacophore search of enzyme inhibitors. The authors also show the use of QC methods for investigation of charge-transfer complexes. 

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