Overview of the Physical State of Proteins Within Cells

The word protein comes from the Greek word proteios, meaning primary. And, indeed, proteins are of primary importance in the study of cell function. It is difficult to imagine a cellular function not linked with proteins. Almost all biochemical catalysis is carried out by protein enzymes. Proteins participate in gene regulation, transcription, and translation. Intracellular filaments give shape to a cell while extracellular proteins hold cells together to form organs. Proteins transport other molecules, such as oxygen, to tissues. Antibody molecules contribute to host defense against infections. Protein hormones relay information between cells. Moreover, protein machines, such as actin-myosin complexes, can perform useful work including cell movement. Thus, studying proteins is a prerequisite in understanding cell structure and function.

The physical characterization of proteins began well over 150 years ago with Mulder’s characterization of the atomic composition of proteins. In the latter half of the nineteenth century Hoppe-Seyler (1864) crystallized hemoglobin and Kühn (1876) purified trypsin. A variety of physical methods have been developed over the years to increase convenience and precision in the characterization and isolation of proteins. These include ultracentrifugation, chromatography, electrophoresis, and others. In many instances our understanding of cell proteins parallels the introduction and use of new techniques to examine their structure and function.

All proteins are constructed as a linear sequence(s) of various numbers and combinations of ∼20 α-amino acids joined by peptide bonds to form structures from thousands to millions of daltons in size. Proteins are the most complex and heterogeneous molecules found in cells, where they account for >50% of the dry weight of cells and ∼75% of tissues. Proteins can be classified into three broad groups: globular, fibrous, and transmembrane (Table 1, Figure 1). Globular proteins are, by definition, globe-shaped, although in practice they can be spherical or ellipsoidal. Globular proteins are generally soluble in aqueous environments. Examples of globular proteins are hemoglobin, serum albumin, and most enzymes. Fibrous proteins are elongated linear molecules that are generally insoluble in water and resist applied stresses and strains. Collagen is a physically tough molecule of connective tissue. Just as collagen gives strength to connective tissues, intermediate filaments linked to desmosomes give strength to cells in tissues. The third general class of proteins, transmembrane proteins, contain a hydrophobic sequence buried within the membrane. These protein categories are not mutually exclusive. For example, the nominally fibrous intermediate filament proteins also have globular domains. Similarly, transmembrane proteins almost always possess globular domains. Thus, these definitions serve as a useful guide but should not be rigidly applied.

Figure 1 General classifications of proteins.

In these schematic representations of globular, fibrous, and transmembrane proteins, hydrophobic regions are shaded. Note that the disposition of hydrophobic residues often reflects the protein class.

Table 1 Broad Classifications for Proteins

A key physical feature of proteins is their hydropathy pattern (i.e., the distribution of hydrophobic and hydrophilic amino acid residues). Indeed, hydrophobic interactions provide the primary net free energy required for protein folding. Figure 1 illustrates the disposition of hydrophobic amino acids in proteins. In an intact globular protein, hydrophobic amino acids are generally shielded from the aqueous environment by coalescing at the center of the molecule, with the more hydrophilic residues exposed at its surface. However, the linear arrangement of hydrophobic residues fluctuates in an apparently random fashion. The α helices within globular proteins may express a hydrophobic face oriented toward the center of the protein. Within these helices hydrophobic residues are nonrandomly positioned every three or four amino acids to yield a hydrophobic face. For coiled-coil α helix–containing fibrous proteins, such as tropomyosin and α-keratin, hydrophobic residues at periodic intervals allow close van der Waals contact of the chains and potentiate assembly as hydrophobic residues are removed from the aqueous environment. Secondarily, regularly spaced charged groups can also contribute to the shape of fibrous proteins. Transmembrane proteins provide a rather different physical arrangement of hydrophobic residues in which hydrophobic residues are collected primarily into a series of amino acids that is embedded within a cell membrane. One important means of analyzing the hydropathy of a sequenced protein is a hydropathy plot. In this method, each amino acid residue is assigned a hydropathy value, an ad hoc measure that largely reflects its relative aqueous solubility; these values are plotted after being averaged. The successful interpretation of hydropathy plots depends on the parameters chosen for averaging. The parameters are the number of residues averaged (amino acid interval or “window”) and how many amino acids are skipped when calculating the next average (step size). Using this approach with a window of ∼10 residues, it is often possible to find the positions of hydrophobic residues coalescing near the interior of globular proteins. The method is particularly useful in predicting transmembrane domains of proteins, generally with a window of ∼20 amino acids. To detect the repetitious pattern of coiled-coil fibrous proteins, however, windows smaller than the repeat length would be required.

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