Protein Structure and Biophysical Properties

Key Insight

Proteins are a diverse class of amphoteric biopolymers, with molecular masses ranging from 5 to 20000 kilodaltons, constructed from amino acids. Structural complexity varies greatly, from simple peptides like insulin (5808 Daltons) to large multimeric glycoproteins such as the human von Willebrand factor (20000 kilodaltons), which consists of up to 80 subunits, each 250 kilodaltons. Most proteins typically have molecular masses between 15 and 200 kilodaltons. These molecules are generally compact yet flexible enough to undergo significant conformational changes in response to environmental shifts, substrate binding, or surface adsorption. Protein structure is highly organized into primary, secondary, tertiary, and quaternary levels, each critical for biological function, and can incorporate non-amino acid elements like prosthetic groups in enzymes or heme groups in hemoglobin.

The primary structure is determined by the amino acid sequence, formed during biosynthesis via peptide bonds. Only the L-isomer of the 20 naturally occurring amino acids is found in proteins, with an average molecular mass of 109 Daltons per amino acid, allowing for protein mass estimation. Peptide bonds possess partial double-bond character, restricting rotation and influencing polypeptide chain conformation. Amino acid side chains, which can be charged, polar, or hydrophobic, dictate a protein's biophysical properties. Charged groups determine the net charge based on pH, while hydrophobic side chains play a substantial role in folding. Cysteine residues are unique for forming reversible disulfide bonds (intramolecular or intermolecular), which stabilize folded structures and can lead to covalently bonded multimeric protein structures. These bonds' reversibility is exploited in techniques like SDS-PAGE and covalent chromatography, the latter used for separating IgG heavy and light chains. Proline, a cyclic imino acid, impacts polypeptide conformation through its cis/trans isomeric forms, and its slow interconversion can limit protein folding rates.

Protein secondary structure elements include alpha helices, beta sheets, and loops. Alpha helices are spiral arrangements of 3.6 amino acid residues per turn, stabilized by intramolecular hydrogen bonds, and can be hydrophobic (e.g., citrate synthase), hydrophilic (e.g., troponin C), or amphipathic (e.g., alcohol dehydrogenase) depending on the amino acid sequence. Beta sheets are very stable planar structures formed by extensive hydrogen bonding (each bond contributes about 1 kilojoule per mole), occurring in parallel, anti-parallel, or mixed configurations. Their formation is often observed during irreversible protein aggregation, requiring vigorous denaturing agents like high concentrations of urea to disrupt. Amyloid proteins and fibers contain numerous beta sheets, explaining their aggregation-prone nature. Loops are flexible regions that connect other secondary structure elements and are crucial for the stability of artificial proteins like single-chain antibodies. Spectroscopic methods, including circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectroscopy, are used to measure secondary structure content and study conformational changes, with CD being an essential tool for monitoring protein integrity, refolding, and thermally or chemically induced unfolding.