Protein noncovalent forces

Noncovalent Forces Stabilizing Protein Structure. Much of protein engineering concerns attempts to alter the stmcture or function of a protein in a predefined way. An understanding of the underlying physicochemical forces that participate in protein folding and stmctural stabilization is thus important. 

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

If the protein of interest is a heteromultimer (composed of more than one type of polypeptide chain), then the protein must be dissociated and its component polypeptide subunits must be separated from one another and sequenced individually. Subunit associations in multimeric proteins are typically maintained solely by noncovalent forces, and therefore most multimeric proteins can usually be dissociated by exposure to pEI extremes, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All of these treatments disrupt polar interactions such as hydrogen bonds both within the protein molecule and between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or charge. Occasionally, heteromultimers are linked together by interchain S—S bridges. In such instances, these cross-links must be cleaved prior to dissociation and isolation of the individual chains. The methods described under step 2 are applicable for this purpose. 

The native, biologically active form of a protein molecule is held together by a delicate balance of noncovalent forces hydrophobic, ionic, van der Waals interactions, and hydrogen bonds. In addition,... 

The complex hierarchy of native protein structure may be disrupted by multiple possible destabilizing mechanisms. As has been described in the foregoing, these processes may disrupt noncovalent forces of interaction or may involve covalent bond breakage or formation. A summary of the processes involved in the irreversible inactivation of proteins is illustrated in Fig. 3 and described briefly in the following section. Detailed discussions of mechanisms of protein desta-... 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

Most proteins contain more than one polypeptide chain. The manner in which these chains associate determines quaternary structure. Binding involves the same types of noncovalent forces mentioned for tertiary structure van der Waals forces, hydrophobic and hydrophilic attractions, and hydrogen bonding. However, the interactions are now interchain rather than infrachain (tertiary structure determination). The quaternary structure of hemoglobin (four almost identical subunits) will be discussed in Chapter 4, that of superoxide dismutase (two identical subunits) will be discussed in Chapter 5, and that of nitrogenase (multiple dissimilar subunits) will be discussed in Chapter 6. 

Cosolvent and temperature effects on various types of noncovalent forces involved in protein-protein interactions are now well documented. These effects have been intensively studied in Douzou s laboratory through their impact on protein fractionation (Douzou and Balny, 1978). Mixed solvents at carefully controlled concentration and temperature variations in the range of normal and subzero temperatures ap-... 

Increased electronegativity causes the disruption Selective precipita-of the noncovalent forces. tion of the proteins... 

Figure 6.1. The role of HMW subunits in gluten structure and functionality. Amino acid sequences derived from direct analysis of purified proteins and the isolation and sequencing of corresponding genes show that the proteins have highly conserved structures, with repetitive domains flanked by shorter nonrepetitive domains containing cysteine residues (SH) available for formation of interchain disulphide bonds. Molecular modelling indicates that the individual repetitive domains form a loose spiral structure (bottom right) while SPM shows that they interact by noncovalent forces to form fibrils (centre right). Includes figures from Parchment et al. (2001) and Humphries et al. (2000).

Noncovalent interactions are central to many key biological functions. The self-assembly of the plasma and organelle membranes, association between receptors and ligands, and folding of RNA and protein molecules are all controlled by noncovalent interactions. Noncovalent forces in the design of proteins have proved useful in gaining insight into... 

Protein tertiary (111°) structure The overall three-dimensional structure of a single polypeptide chain, including positions of disulfide bonds. Noncovalent forces such as hydrogen bonding, electrostatic forces, and hydrophobic effects are also important. 

Protein structure is typically classified as consisting of four levels primary (1°), secondary (11°), tertiary (III°), and quaternary (IV°). Primary structure is the sequence of amino acids in the protein. Secondary structure is the local three-dimensional spatial arrangement of amino acids that are close to one another in the primary sequence. a-Helices and P-sheets compose the majority of secondary structures in all known proteins. Tertiary structure is the spatial arrangement of amino acid residues that are far apart in the linear primary sequence of a single polypeptide chain, and it includes disulfide bonds and noncovalent forces. These noncovalent forces include hydrogen bonding, which is also the primary stabilization force for the formation of a-helices and P-sheets, electrostatic interactions, van der Waals forces, and hydrophobic effects. Quaternary structure is the manner in which subunits of a multi-subunit protein are arranged with respect to one another. 

---