Protein CSPs mechanisms

This methodology was similarly applied to cold shock proteins (CSP) [81]. They are a family of small single domain proteins, with a highly conserved sequence identity [82,83]. The known structures of CSPs consist of a secondary structure of two amphipathic /3-sheets. The first /3-sheet is formed by three antiparallel /3-strands and the second contains two anti-parallel /3-strands. These two /3-sheets form a hydrophobic core and a predominantly hydrophilic surface [84]. Many mutational forms of these proteins have been studied experimentally and hence they can be used as a model to study the effects of mutations on folding stability and mechanisms. 

Fig. 7. Conservation of the unfolding/folding mechanism of cold-shock proteins (Csp) from B. subtilis (Bs), B. caldolyticus (fid), and Thermotoga maritima (Tm). (a) Equilibrium unfolding transitions of Csp from Bs (A), Be ( ), and Tm ( ) induced by GdmCI at 25° and monitored by intrinsic fluorescence. Least-squares fit analyses based on the two-state model yield stabilization energies AGstab = 11.3,20.1, and 26.2 kJ/mol for Csp from Bs, Be, and Tm, respectively, (b) Kinetics of unfolding (open symbols) and refolding (closed symbols) of Bs (A, A), Be ( , ) and T Csp (O, ), respectively. The apparent rate constants, X, are plotted against the GdmCI concentration. The fits are on the basis of the linear two-state model. ...

Different classifications for the chiral CSPs have been described. They are based on the chemical structure of the chiral selectors and on the chiral recognition mechanism involved. In this chapter we will use a classification based mainly on the chemical structure of the selectors. The selectors are classified in three groups (i) CSPs with low-molecular-weight selectors, such as Pirkle type CSPs, ionic and ligand exchange CSPs, (ii) CSPs with macrocyclic selectors, such as CDs, crown-ethers and macrocyclic antibiotics, and (iii) CSPs with macromolecular selectors, such as polysaccharides, synthetic polymers, molecular imprinted polymers and proteins. These different types of CSPs, frequently used for the analysis of chiral pharmaceuticals, are discussed in more detail later. 

Binding of hydrophobic molecules by specific protein carriers appears to be a very efficient mechanism to increase both solubility and transport of these molecular messengers in a hydrophilic medium. OBPs and CSPs may represent a successful application of this principle. In particular, the molecular mechanisms of transport of hydrophobic molecules may be more ancient than that most ancient of senses, olfaction. The olfactory system may have developed to extract the hydrophobic odorants from the air environment and optimize their transport and delivery to sensory cells. 

HPLC-CSPs are based on molecules of known stereochemical composition immobilized on liquid chromatographic supports. Single enantiomorphs, diastereomers, diastereomeric mixtures, and chiral polymers (such as proteins) have been used as the chiral selector. The chiral recognition mechanisms operating on these phases are the result of the formation of temporary diastereomeric complexes between the enantiomeric solute molecules and immobilized chiral selector. The difference in energy between the resulting diastereomeric solute/CSP complexes determines the magnitude of the observed stereoselectivity, whereas the sum total of the interactions between the solute and CSP chiral and achiral, determines the observed retention and efficiency. 

The binding interactions between the solute and protein usually involves stereospedfic and nonstereospecific mechanisms. These mechanisms make the type V CSPs sensitive to the composition of the mobile phase, temperature, flow rate, and pH. These parameters can be adjusted to improve the chromatography and stereoselectivity of specific solutes on the AGP CSP (88,89), OVM CSP (90,91), BSA CSP (92,93), and HSA CSP (94). 

The use of chiral stationary phases (CSP) in liquid chromatography continues to grow at an impressive rate. These CSPs contain natural materials such as cellulose and starch as well as totally synthetic materials, utilizing enantioselective and retentive mechanisms ranging from inclusion complexation to Ti-electron interactions. The major structural features found in chiral stationary phases include cellulose, starch, cyclodextrins, synthetic polymers, proteins, crown ethers, metal complexes, and aromatic w-electron systems. 

The accurate determination of the adsorption isotherm parameters of the two enantiomers on a CSP is of fundamental importance to do computer-assisted optimization to scale up the process. Such determinations are usually done with an analytical column and the most traditional method to determine the parameters and saturation capacity is by frontal analysis (see section 3.4.2). The aim of paper III was to investigate the adsorption behavior and the chiral capacity of the newly developed Kromasil CHI-TBB column using a typical model compound. Many of the previous studies from the group have been made on low-capacity protein columns which has revealed interesting information about the separation mechanism [103, 110, 111], For this reason a column really aimed for preparative chiral separations was chosen for investigation [134], As solute the enantiomers of 2-phenylbutyric acid was chosen. 

Most chiral HPLC analyses are performed on CSPs. General classification of CSPs and rules for which columns may be most appropriate for a given separation, based on solute structure, have been described in detail elsewhere. Nominally, CSPs fall into four primary categories (there are additional lesser used approaches) donor-acceptor (Pirkle) type, polymer-based carbohydrates, inclusion complexation type, and protein based. Examples of each CSP type, along with the proposed chiral recognition mechanism, analyte requirement(s), and mode of operation, are given in Table 3. Normal-phase operation indicates that solute elution is promoted by the addition of polar solvent, whereas in reversed-phase operation elution is promoted by a decrease in mobile-phase polarity. 

It is true that the unambiguous elucidation of chiral recognition mechanisms on various protein-based CSPs is challenging and often difficult since precise information about the tertiary and quaternary stmctures of proteins is not always available. Multiple stereo-specific sites may be involved in chiral recognition process. However, it is encouraging to see the progresses that have been made in this field in recent years [17, 95—102]. 

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