Protein three-dimensional conformation

Knowledge and understanding of protein structure and properties in the 1950 s was rapidly evolving. The unique secondary, tertiary, and even quaternary structures of proteins were becoming understood6 8) and the delicateness of protein three-dimensional conformation was recognized, including the possibility for denatura-tion at liquid/air and solid/liquid interfaces x 3). 

This thiol-disulfide interconversion is a key part of numerous biological processes. WeTJ see in Chapter 26, for instance, that disulfide formation is involved in defining the structure and three-dimensional conformations of proteins, where disulfide "bridges" often form cross-links between q steine amino acid units in the protein chains. Disulfide formation is also involved in the process by which cells protect themselves from oxidative degradation. A cellular component called glutathione removes potentially harmful oxidants and is itself oxidized to glutathione disulfide in the process. Reduction back to the thiol requires the coenzyme flavin adenine dinucleotide (reduced), abbreviated FADH2. 

The three-dimensional conformation of a protein is called its tertiary structure. An a-helix can be either twisted, folded, or folded and twisted into a definite geometric pattern. These structures are stabilized by dispersion forces, hydrogen bonding, and other intermo-lecular forces. 

The term tertiary stmcmre refers to the entire three-dimensional conformation of a polypeptide. It indicates, in three-dimensional space, how secondary stmcmral feamres—hehces, sheets, bends, mrns, and loops— assemble to form domains and how these domains relate spatially to one another. A domain is a section of protein strucmre sufficient to perform a particular chemical or physical task such as binding of a substrate... 

Enzymes are proteins, i.e. sequences of amino acids linked by peptide bonds. The sequence of amino acids within the polypeptide chain is characteristic of each enzyme. This leads to a specific three-dimensional conformation for each enzyme in which the molecular chains are folded in such a way that certain key amino acids are situated in specific strategic locations. This folded arrangement, together with the positioning of key amino acids, gives rise to the remarkable catalytic activity associated with enzymes. 

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

Phosphate is charged (2—) so when it is incorporated into an enzyme, alterations in the electrostatic attractions between parts of the enzyme molecule will occur causing a change in the three-dimensional conformation of the protein. The effect may be to expose the active site to allow substrate binding (if phosphorylation activates the enzyme) or may hide the active site, so switching off the enzyme. 

In addition to identifying protein partners, yeast two-hybrid technology can be used to identify and study in detail the interaction domains between two proteins. Here, bait and/or fish truncation or deletion constructs of the parent proteins are engineered and characterized as described earlier (see 3.1 Selection and characterization of bait constructs). These are then investigated for association in a yeast two-hybrid interaction assay. Once the BD has been identified, it can be further refined by mutagenesis. The same caveat applies to these studies as for the identification of associating proteins, i.e., it is assumed that the respective fusion proteins fold and adopt the same or a similar three-dimensional conformation to the native protein. This is not always the case and results should be interpreted with caution and if possible, always validated by an alternative experimental approach. O Table 19-1 shows an example of mapping the... 

As proteins emerge from ribosomes, they fold into three-dimensional conformations that are essential for their subsequent biologic activity. Generally, four levels of protein shape are distinguished ... 

The three-dimensional conformation of a protein, made up of secondary structural elements and unordered sections, is referred to... 

The actions of enzymes as catalysts depend on their three-dimensional conformation, or how the protein is folded into a three-dimensional object. A protein is said to be denatured if its three-dimensional conformation is altered, such as by heat or mechanical stirring, and is no longer biochemically active as a catalyst. 

Most proteins must be folded into a specific three-dimensional conformation to express their specificity and activities, which comphcates the DSP [212]. Researchers in the area of RME of proteins/enzymes have reafized this and directed more efforts in developing novel and imaginative techniques in RME as well as coupling the existing techniques such as chromatography, electrophoresis, and membrane extractions with RME. Such promising techniques developed in the recent past have been discussed in this section. Apart from these techniques, use of novel surfactants in the RME and surfactant based separation processes (e.g., cloud-point extraction) are also considered. 

Anfinsen and White concluded that the three-dimensional conformation of proteins is specified by their amino acid sequence. 

Typically, proteins fold to organize a very specific globular conformation, known as the protein s native state, which is in general reasonably stable and unique. It is this well-defined three-dimensional conformation of a polypeptide chain that determines the macroscopic properties and function of a protein. The folding mechanism and biological functionality are directly related to the polypeptide sequence a completely random amino acid sequence is unlikely to form a functional structure. In this view, polypeptide sequence... 

The native three-dimensional conformation of a protein is maintained by a range of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulfide bonds), in addition to the peptide bonds between individual amino acids. 

The tertiary structure of a protein is its complete three-dimensional conformation. Think of the secondary structure as a spatial pattern in a local region of the molecule. Parts of the protein may have the a-helical structure, while other parts may have the pleated-sheet structure, and still other parts may be random coils. The tertiary structure includes all the secondary structure and all the kinks and folds in between. The tertiary structure of a typical globular protein is represented in Figure 24-17. 

X-ray crystallographers have now determined the structures of approximately one hundred biological macromolecules — proteins, nucleic acids, and viruses — to atomic resolution. These investigations have demonstrated that, unlike synthetic polymers, the biological molecules have specific three-dimensional conformations. Indeed, all information required to specify the structure of a protein is contained in the sequence of amino acids, and therefore the structure is also implicit in the sequence of nucleotides in the DNA or RNA genome. Analysis of the structures has provided explanations of their biological functions, and has revealed that there are recurrent architectural themes in their de-sign (J, 2). 

The peptide linkages between amino acids form the primary structure of the protein. The primary structure is all that the nucleotide sequence of the genetic material determines, and therefore the primary structure contains all information necessary to specify the complete three-dimensional conformation. 

The biological activity of proteins generally depends on a unique three-dimensional conformation, which in turn is inherently linked to its primary sequence. Protein folding, the conversion of the translated polypeptide chain into the native state of a protein, is the critical link between gene sequence and three-dimensional structure. Mechanistically, folding is believed to proceed through a predetermined and ordered pathway, either via kinetic intermediates or by direct transition from the unfolded to the native state [99]. In both cases, local and non-local interactions alike stabilize transient structures along the pathway and funnel the intermediates towards the native state. 

---