Protein structure - Pharmaceutical Biotechnology: Concepts and Aapplications - page 30

Disulfi de bonds represent the major covalent bond type that can help stabilize a polypep-
tide's native three-dimensional structure. Intracellular proteins, although generally harbouring
multiple cysteine residues, rarely form disulfi de linkages, due to the reducing environment
that prevails within the cell. Extracellular proteins, in contrast, are usually exposed to a more
oxidizing environment, conducive to disulfi de bond formation. In many cases the reduction
(i.e. breaking) of disulfi de linkages has little effect upon a polypeptide's native conformation.
However, in other cases (particularly disulfi de-rich proteins), disruption of this covalent link-
age does render the protein less conformationally s table . In these cases the disulfi de linkages
likely serve to 'lock' functional/structurally important elements of domain/tertiary structure
in place.
The description of protein structure as presented thus far may lead to the conclusion that
proteins are static, rigid structures. This is not the case. A protein's constituent atoms are con-
stantly in motion, and groups ranging from individual amino acid side chains to entire domains
can be displaced via random motion by anything up to approximately 0.2 nm. A protein's
conformation, therefore, displays a limited degree of fl exibility, and such movement is termed
'breathing'.
Breathing can sometimes be functionally signifi cant by, for example, allowing small molecules
to diffuse in/out if the protein's interior. In addition to breathing, some proteins may undergo more
marked (usually reversible) conformational changes. Such changes are usually functionally sig-
nifi cant. Most often they are induced by biospecifi c ligand interactions (e.g. binding of a substrate
to an enzyme or antigen binding to an antibody).

2.4.1 Structural prediction

Currently, there exists an enormous and growing defi cit between the number of polypeptides
whose amino acid sequence has been determined and the numbers of polypeptides whose three-
dimensional structure has been resolved. Given the complexities of resolving three-dimensional
structure experimentally, it is not surprising that scientists are continually attempting to develop
methods by which they could predict higher order structure from amino acid sequence data.
Although modestly successful secondary structure predictive approaches have been developed,
no method by which tertiary structure may be predicted from primary data has thus far been
developed.
Over 20 different methods of secondary structure prediction have been reported ( Table 2.6).
The approaches taken fall into two main categories:

Table 2.6 Some secondary structure predictive methods currently used. Refer to
text for further details

Method Basis of prediction

Chou and Fasman Empirical statistical method

Garnier, Osguthorpe and Robson (GOR) Empirical statistical method

EMBL profi le neural network (PHD) Empirical statistical method

Protein sequence analysis (PSA) Empirical statistical method

Lim Physicochemical criteria