Riassunto esame Biochemistry, Prof. Palese Luigi Leonardo, libro consigliato Biochimica, Voet

Biochemistry

Professor L.L. Palese

Academic year 2025/2026

Part II: biomolecules

Solubilities of proteins

Different proteins vary greatly in their solubilities under a given set of conditions: certain proteins precipitate from solution under conditions in which others remain quite soluble. This effect is routinely used as a basis for protein purification.

Solubility is one of the most important properties for protein: the quantity of a chemical species that we can put in a solution before precipitation occurs. Purified proteins can be solubilized until a certain concentration.

Effect of salt concentrations

The solubility of a protein in aqueous solution is a sensitive function of the concentrations of dissolved salts. At low ionic strength it generally increases with the salt concentration, a phenomenon known as salting in: proteins, which have multiple inherent ionic charges, are prevented from forming aggregates and thus prevented from precipitating by the ions of the salt that shield their charges and thereby increase the protein’s solubility. At high ionic strength, instead, proteins’ solubility decreases as salt concentration increases, a phenomenon known as salting out: salt concentration is that high that molecules of solvent are almost completely occupied in solvating its ions and become insufficient to dissolve other solutes, therefore proteins form aggregates and precipitate.

Effect of organic solvents

Water-miscible organic solvents, such as acetone and ethanol, are generally good protein precipitants because their low dielectric constants reduce the solvating power of their aqueous solutions for dissolved ions such as proteins.

Effects of pH

Proteins generally bear numerous ionizable groups that have a variety of pK's. At a pH characteristic for each protein, the isoelectric point, the positive charges on the molecule exactly balance its negative charges and the protein molecule carries no net charge and is therefore immobile in an electric field.

Physicochemical considerations suggest that the solubility properties of uncharged molecules are insensitive to the salt concentration. To a first approximation, therefore, a protein at its isoelectric point should not be subject to salting in. Conversely, as the pH is varied and thus as the protein's net charge increases, it should be increasingly subject to salting in because the electrostatic interactions between neighbouring molecules that promote aggregation and precipitation should likewise increase.

Hence, in solutions of moderate salt concentrations, the solubility of a protein as a function of pH is expected to be at a minimum at the protein's isoelectric point and to increase about this point with respect to pH.

Secondary structure of proteins

A polymer’s secondary structure is defined as the local conformation of its backbone: L. Pauling and R. Corey determined the X-ray structures of several amino acids and dipeptide and indicated that the peptide group has a rigid, planar structure which is a consequence of resonance interactions that give the peptide bond an almost 40% double-bond character.

Peptide groups usually assume the trans conformations: successive alpha-carbons are on opposite sides of the peptide bond joining them. The backbone of a protein is a linked sequence of rigid planar peptide groups and therefore we can specify a polypeptide’s backbone conformation by the torsion angles about the C_α-N bond (φ) and the C_α-C bond (ψ) of each of its amino acid residues, both 180 when the polypeptide is in its planar, fully extended conformation. The sterically allowed values of ψ and φ can be determined by calculating the distances between the atoms of a tripeptide at all values of ψ and φ for the central peptide unit. Sterically forbidden conformations are those in which any nonbonding interatomic distance is less than its corresponding Van der Waals distance. Such information is summarized in a conformation map or Ramachandran diagram, only three small regions of the conformational map are physically accessible to a polypeptide chain.

Helices

If a polypeptide chain is twisted by the same amount about each of its alpha-carbons, it assumes a helical conformation, held in place by hydrogen bonds. Among all, only one helical polypeptide conformation has simultaneously allowed conformation angles and a favorable hydrogen bonding pattern: the α helix, a particular arrangement of the polypeptide chain characterized by φ = -57 and ψ = -47 and the presence of 3.6 amino acids per turn. It is a common secondary structural element of both fibrous and globular proteins.

Certain synthetic homopolypeptides assume conformations that are models for helices in particular proteins: polyproline is unable to assume any common secondary structure due to the conformational constraints imposed by its cyclic lateral chain and the lack of hydrogens in its backbone prevent it from being stabilized by hydrogen bondings. However, under proper conditions polyproline precipitates from solution as left-handed helix of all-trans peptides that has 3 residues per turn. This rather extended conformation, known as polyproline II helix, permits the proline side chains to avoid each other. Also polyglycine precipitates from solution as a helix whose parameters are essentially identical to those of polyproline, even if they are the most and the least conformationally constrained polypeptides. Polyglycine and polyproline helices are of biological interest because they form the basic structural motif of collagen, a structural protein.

Beta structures

Pauling and Corey also postulated the existence of a different polypeptide secondary structure, the β-pleated sheet, which utilizes the full hydrogen bonding capacity of the polypeptide backbone. In this case, hydrogen bonding occurs between neighbouring polypeptide chains rather than within one as in α helices. β-pleated sheets come in two varieties: the antiparallel and the parallel ones.

Nonrepetitive structures

Globular proteins consist of, on average, 31% α helix and 28% β-pleated sheet. The remaining polypeptide segments are said to have a coil or loop conformation, irregular and hence more difficult to describe. We must not confuse them with the random coils, which refers to the totally disordered and rapidly fluctuating set of conformation assumed by denatured proteins and other polymers in solution. Globular proteins consist largely of approximately straight runs of secondary structures joined by stretches of polypeptide that abruptly change direction. Such reverse turns or β bends almost always occur at protein surfaces.

Tertiary structure of proteins

The tertiary structure of a protein is its 3D arrangement of its 2° structural elements, together with the spatial dispositions of its side chains.

Protein stability

Native proteins are only marginally stable under physiological conditions. The energy required to denature a protein is remarkably small, roughly 0.4 kJ/mol per amino acid. For a typical protein of 100 residues, the total stability is only about 40 kJ/mol, which is equivalent to the strength of just two average hydrogen bonds. Protein structure is not held together by one massive force, but rather by a delicate balance of powerful, opposing noncovalent interactions, these include electrostatic interactions, attraction and repulsion, hydrogen bonding, internal and with water, hydrophobic forces. While the net stability is low, the individual forces involved are massive, often totaling thousands of kilojoules per mole. The final structure exists only because these "countervailing forces" almost perfectly cancel each other out.

• Electrostatic forces
  • Ionic interactions: the association of two ionic protein groups of opposite charge is known as an ion pair or salt bridge. Free ions in aqueous solution are highly solvated, and the formation of a salt bridge has the entropic penalty of localizing the salt bridge’s charged side chains. Consequently, the free energy of solvation of two separated ions is about equal to the free energy of formation of their unsolvated ion pair. Ion pairs therefore contribute little stability toward a protein’s native structure.
  • Dipole–dipole interactions: the noncovalent associations between electrically neutral molecules, van der Waals forces, arise from electrostatic interactions among permanent and/or induced dipoles. Even if these interactions are generally much weaker than the interactions of ion pairs and vary with r^-3, so they rapidly attenuate with distance. Interactions among permanent dipoles, in the low dielectric constant core of a protein, are important structural determinants in proteins. Although nonpolar molecules are electrically neutral, at any instant they have a small dipole moment resulting from the rapid fluctuating motions of their electrons. This transient dipole moment polarizes the electrons in a neighboring group, giving rise to a dipole moment. These London dispersion forces are extremely weak and are only significant for contacting groups because their association energy is proportional to r^-6. Nevertheless, the great numbers of interatomic contacts in the closely packed interiors of proteins make London forces a major influence in determining their conformations.
• Hydrogen bonding: Hydrogen bonds are predominantly electrostatic interactions, with 10% covalent character, between a weakly acidic donor group and an acceptor that bears a lone pair of electrons. Hydrogen bonds have association energies that are normally between those for covalent bonds and van der Waals forces. Hydrogen bonds, H bonds, are much more directional than are van der Waals forces but less so than are covalent bonds. While hydrogen bonds are a defining feature of protein structures, their actual contribution to the overall stability of the native state is not so important: an unfolded protein in an aqueous environment forms an almost equivalent number of hydrogen bonds with the surrounding water molecules. Since the free energy of stabilization is defined as the difference between the folded and unfolded states, this energetic competition with the solvent suggests that hydrogen bonds might provide little to no net stability. However, several factors ensure that internal hydrogen bonding remains energetically favorable. First, because hydrogen bonds are essentially electrostatic, they become significantly stronger in the low-polarity environment of a protein's interior compared to the high-polarity aqueous solvent. Furthermore, an entropic advantage arises because water molecules bound to an unfolded polypeptide are highly constrained in their orientation. When the protein folds and forms internal hydrogen bonds, these water molecules are released into the bulk solvent, increasing the overall entropy of the system.
• Hydrophobic interactions: while Van der Waals forces are universal electromagnetic attractions that occur between all atoms and molecules, regardless of whether they are polar or nonpolar, the hydrophobic interaction is not a direct attractive force between nonpolar molecules; rather, it is a thermodynamic phenomenon driven by the properties of the solvent, specifically water. When a nonpolar substance is introduced into an aqueous environment, water molecules are forced to highly organize themselves into cage-like, clathrates, structures around the solute to maintain their hydrogen-bonding network. Because this high degree of organization leads to a decrease in entropy—which is energetically unfavorable—the system naturally seeks to minimize the surface area of contact between the water and the nonpolar substance. Therefore, the nonpolar molecules are pushed together not because they "like" each other, but because their aggregation allows the water molecules to be excluded and return to a more disordered, high-entropy state. Ultimately, the key distinction lies in their nature: Van der Waals forces are an intrinsic property of matter based on electronic attraction, whereas the hydrophobic effect is an entropy-driven process dictated by the surrounding water.

Quaternary structure

Some proteins are composed of more than one polypeptide chain. The spatial arrangement of these subunits is known as a protein’s quaternary structure. In large assemblies of proteins, such as collagen fibrils, the advantages of subunit construction over the synthesis of one huge polypeptide chain are analogous to those of using prefabricated components in constructing a building: defects can be repaired by simply replacing the damaged subunit rather than the entire protein, the site of subunit manufacture can be different from the site of assembly into the final product, and the only genetic information necessary to specify the entire protein is that specifying its few different self-assembling subunits. A multisubunit protein may consist of identical or non-identical polypeptide chains. We shall refer to proteins with identical subunits as oligomers and to these identical subunits as protomers.

Fibrous proteins

Fibrous proteins are highly elongated molecules whose secondary structures are their dominant structural motifs. Many fibrous proteins function as structural materials that have a protective, connective, or supportive role in living organisms. Others have motive functions. In this section, we shall discuss structure–function relationships in two common and well-characterized fibrous proteins: keratin and collagen. The structural simplicity of these proteins relative to those of globular proteins makes them particularly easy to understand how their structures suit them to their biological roles. Fibrous molecules rarely crystallize and hence are usually not subject to structural determination by single-crystal X-ray structure analysis. Rather than crystallizing, they associate as fibers in which their long molecular axes are more or less parallel to the fiber axis but in which they lack specific orientation in other directions. Consequently, the structures of fibrous proteins are not known in great detail.

α Keratin

Keratin is a mechanically durable and chemically unreactive protein that occurs in all higher vertebrates. Keratins have been classified as either α keratins, which occur in mammals, or β keratins, which occur in birds and reptiles. Mammals have over 50 keratin genes, which are expressed in a tissue-specific manner and whose products are classified as belonging to families of relatively acidic, Type I, and relatively basic, Type II, polypeptides. Keratin filaments, which form the intermediate filaments of skin cells, must contain at least one member of each type.

Electron microscopic studies indicate that hair, which is composed mainly of α keratin, consists of a hierarchy of structures. A typical hair is 20 m in diameter and is constructed from dead cells, each of which contains packed macrofibrils that are oriented parallel to the hair fiber. The macrofibrils are constructed from microfibrils that are cemented together by an amorphous protein matrix of high sulfur content. The X-ray diffraction pattern of keratin resembles that expected for an α helix.

α keratin polypeptides form closely associated pairs of helices in which each pair is composed of a Type I and a Type II keratin chain twisted in parallel into a left-handed coil. This assembly is said to have a coiled coil structure because each helix axis itself follows a helical path. The conformation of keratin’s coiled coil is a consequence of its primary structure. The central 310-residue segment of each polypeptide chain has a heptad, 7-residues, pseudo repeat, a-b-c-d-e-f-g, with nonpolar residues predominating at positions a and d. Since an α helix has 3.6 residues per turn, keratin’s a and d residues line up on one side of the helix to form a hydrophobic strip that promotes its association with a similar strip on another helix.

α Keratin is rich in Cys residues, which form disulfide bonds that cross-link adjacent polypeptide chains. This provides keratin’s insolubility and resistance to stretching, two of its most important biological properties. The keratins are classified as “hard” or “soft” according to whether they have a high or low sulfur content. Hard keratins, such as those of hair, are less bendable than soft keratins, such as those of skin, because the disulfide bonds resist any forces tending to deform them. The disulfide bonds can be reductively cleaved with mercaptans. The springiness of hair and wool fibers is a consequence of the coiled coil’s tendency to untwist when stretched and to recover its original conformation when the external force is relaxed.

Collagen

Collagen occurs in all multicellular animals and is the most abundant protein of vertebrates, comprising 30% of their protein mass. It is an extracellular protein that is organized into insoluble fibers of great tensile strength. This suits collagen to its role as the major stress bearing component of connective tissues such as bone, cartilage and teeth, it is however present in every tissue. Collagen has a distinctive amino acid composition: nearly one-third of its residues are Gly, another 15 to 30% of them are Pro and 4-hydroxyprolyl, Hyp, residues. 3-Hydroxyprolyl and 5-hydroxylysyl, Hyl, residues also occur in collagen but in smaller amounts.

The hydroxylated residues appear after the collagen polypeptides are synthesized, when certain Pro residues are converted to Hyp in a reaction catalyzed by the enzyme prolyl hydroxylase. Hyp confers stability on collagen, possibly through intramolecular hydrogen bonds that involve bridging water molecules. If collagen is synthesized under conditions that inactivate prolyl hydroxylase, it loses its native conformation at 24°C, whereas normal collagen denatures at 39°C, heat-denatured collagen is known as gelatin. Prolyl hydroxylase requires ascorbic acid, vitamin C, to maintain its enzymatic activity. In the vitamin C deficiency disease scurvy, the collagen synthesized cannot form fibers properly. This results in skin lesions, blood vessel fragility and poor wound healing that are symptomatic of this ultimately fatal vitamin deficiency disease.

The amino acid sequence of bovine collagen α1(I), which is similar