Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Proteins
Structure of Globular Proteins

Class="center">Introduction/introduction.files/image021.jpg" width="636"/>

Fig. 10.1.

The Tertiary Structure of a protein refers to the three-dimensional arrangement of all atoms in a single polypeptide chain. Some Proteins consist of multiple polypeptide chains, where each chain Functions as a subunit, or monomer. Dimers contain two polypeptide chains, trimers contain three, and tetramers contain four. Hemoglobin is a classic example of a tetramer, comprising two identical a-chains and two identical ß-chains.

The term Quaternary Cell/13.html">Protein Structure describes the spatial arrangement and interaction of subunits that make up a multi-subunit protein molecule.

X-ray crystallography has successfully resolved the three-dimensional structures of over a hundred different proteins. The coordinates of most atoms, with the exception of hydrogen, can be determined with an accuracy of 0.1 nm. To date, crystallographic studies have focused primarily on Water-soluble Globular proteins; therefore, the following Discussion applies specifically to this class of proteins.

The surface of a protein globule is populated predominantly by polar groups and charged atoms that favorably interact with the aqueous environment. These include the polar backbone groups >N—H and >C=O, the charged side chains of Glu, Asp, Lys⁺, and Arg⁺ residues, as well as the polar side chains of residues such as Ser, Thr, Asn, Gln, and others. Ionic bonds, commonly referred to as salt bridges (Chapter 7), sometimes form on the globule surface between oppositely charged groups (e.g., Glu and Lys⁺). Additionally, the surface features a small number of nonpolar atoms.

The interior of a protein globule forms a nonpolar microenvironment, shielded from contact with the surrounding solvent by the tight packing of atoms. The Hydrophobic core is composed primarily of nonpolar groups belonging to the aliphatic and aromatic side chains of Ala, Val, Ile, Leu, Met, Phe, and Trp. It is energetically unfavorable for polar or charged groups to reside in this hydrophobic milieu unless they can form interactions with another polar group or an oppositely charged atom. Consequently, backbone >N—H and >C=O groups sequestered within the interior form Hydrogen Bonds with one another, resulting in the generation of a-helices and ß-sheets. Similarly, oppositely charged groups buried inside the globule (such as Glu and Lys⁺) form ionic bonds known as salt bridges.

A disulfide bridge is a covalent bond formed between two Cysteine residues. Such crosslinks are characteristic of certain secretory proteins (Chapter 6) and may be located either in the interior of the globule or on its surface. Many proteins lack Disulfide Bonds entirely, even when reduced cysteines are present.

Renaturation experiments demonstrate that denatured biologically active proteins can spontaneously refold into their native conformation and recover full activity. This indicates that under physiological conditions, the native three-dimensional structure of a protein is thermodynamically stable, corresponding to a free-energy minimum. Furthermore, these experiments reveal that the information required for proper protein folding is entirely encoded within its Amino Acid Sequence.

In principle, therefore, the three-dimensional structure of any protein can be predicted theoretically from its amino acid sequence. This approach is especially valuable when crystallographic determination of a molecular conformation is not feasible. Although the accuracy of Secondary structure prediction (Chapter 9) is now quite high, predicting tertiary structure remains one of the major unsolved problems in molecular biology.

The folding of a polypeptide chain from an extended state must proceed via a single pathway or a very limited number of routes. Consider a protein molecule composed of 50 residues, each capable of adopting 10 distinct Conformations. The total number of possible conformations would be 10⁵⁰; given a characteristic molecular rearrangement time of 10^-13 s, sampling each conformation even once would take approximately 10³⁷ s (~10³⁰ years). Consequently, there must be a directed folding pathway that drastically restricts this conformational search. One leading hypothesis suggests that specific local segments of the protein, such as a-helices, form first and act as nucleation centers for the Condensation of the rest of the molecule.

The stability of folded protein molecules in an aqueous environment is remarkably low: for hen egg-white Lysozyme, it is approximately 40 kJ/mol. The primary driving force behind folding is the entropic hydrophobic effect (Chapter 7), which compels nonpolar groups to escape the aqueous solvent and sequester within the core of the globule. The structure is further stabilized by The formation of internal hydrogen bonds, disulfide bridges, and salt bridges. Conversely, an opposing entropic effect hinders folding. This stems from the fact that the folded state restricts the conformational freedom of both the backbone and side chains compared to the unfolded state; a reduction in conformational Entropy is thermodynamically unfavorable.

Within a folded protein globule, certain polypeptide segments form a-helices or ß-structures, while others adopt irregular yet well-defined conformations (coil regions). The polypeptide chain folds into a remarkably complex architecture, as illustrated in Fig. 10.2, which depicts the lysozyme molecule as a series of segments connecting successive Ca-atoms. (Compare this representation with the simplified model shown in Fig. 10.1).

Domains: During the folding of large polypeptide chains, two or more spatially distinct regions known as domains frequently form. Structurally, each domain resembles a small, independent protein. A single domain typically contains 40 to 300 residues. In the immunoglobulin molecule (Chapter 40), both the heavy and light chains comprise multiple domains; one of these, the constant domain of the light chain, is illustrated in Fig. 10.1.

Fig. 10.2.

All domains can be classified into four major categories: a/a, ß/ß, a/ß, and a + ß, depending on the relative Spatial Organization of their a-helical and ß-structural segments. Examples of domains from each class are shown in Fig. 10.1; polypeptide chains are represented as ribbons, with a-helices depicted as spiral coils, ß-structures as arrows, and irregular regions as light loops. Side chains are omitted for clarity, although in actual proteins, the space between backbone atoms is densely packed with side-chain atoms. a/a-Domains consist predominantly of a-helices, with virtually no ß-Structure. Their a-helices are packed in a manner that keeps nonpolar side chains buried in the interior. ß/ß-Domains contain several ß-sheets and little to no a-helix content. This class is exemplified in the diagram by the constant domain of an immunoglobulin molecule, which consists of two tightly packed ß-sheets. As seen in the diagram, these ß-sheets are not flat but slightly twisted. In a/ß-domains, a- and ß-segments alternate along the chain, frequently forming a parallel ß-sheet surrounded by a-helices. In a + ß-domains, the a- and ß-regions are generally segregated into distinct segments of the polypeptide chain.

The assembly of subunits in multimeric (multi-subunit) proteins is governed by the same types of noncovalent interactions responsible for tertiary structure formation. Subunits are typically packed with a high degree of Symmetry (though not always strictly symmetrical), as seen in hemoglobin, where the four subunits are arranged at the vertices of a tetrahedron (Chapter 15).

Internal Dynamics of Proteins. Experimental evidence demonstrates that proteins are not rigid structures; their constituent parts undergo continuous motion relative to one another, reflecting the internal dynamics of proteins. In many globular proteins, including the four examples shown in Fig. 10.1, backbone atoms can fluctuate by about 0.01 nm around their average positions, while surface side chains exhibit even greater mobility.