Electrostatic interactions

Electrostatic interactions can be either charge-charge interactions between charged species (ionic interactions) or dipole-dipole type interactions between electrically neutral species (van der Waals forces) (Voet and Voet, 2004). In proteins, the charged carboxylate groups of acidic (Glu, Asp) and amino groups of basic (Lys, Arg, His) residues can participate in specific ionic interactions with each other, forming salt bridges (or ion pairs). These interactions occur at distances of the charged species from each other of ≤ 4 Å, and can additionally result in hydrogen bonds if the species are closer, at about ≤ 3.5 Å. Salt bridges are the strongest of the forces stabilising proteins. Their contribution to protein stability depends upon their geometry, including how the side chains are positioned relative to one another, and where they are located in the protein itself. The electrostatic potential between two charges varies with the distance between

the two and the dielectric constant of the medium they are in, as described by Coulomb’s law:

ࢁ = ࢑

ࢿ࢘࢏࢐ Equation 1-2

where k is a conversion factor to the desired energy units, qi and qj are the two

charges, ε is the dielectric constant of the medium and r is the distance between the charges. The equation shows that the attraction between the charges is higher in a low dielectric medium such as the interior of a protein, where the dielectric constant is estimated to be between 3 and 5, depending on the degree of flexibility of the polypeptide backbone (Honig et al., 1986). Estimations of the number of ion pairs buried in the interior of proteins range from 17 % (Barlow and Thornton, 1983) to approximately 33% (Kumar and Nussinov, 1999) to 53 % (Lesser and Rose, 1990). Salt bridges can be stabilising or destabilising in proteins because of the balance between the favourable charge-charge interactions and the unfavourable loss of solvation and structural ordering (Bosshard et al., 2004). This was shown quite neatly with a study of the conformers in nuclear magnetic resonance (NMR) structures of proteins, where ion pairs were lost in some conformers and other ion pairs were gained, with some of the interactions being stabilising and others destabilising (Kumar and Nussinov, 2001). This study also illustrated that the networks of interactions within proteins are in constant dynamic equilibrium.

The net contribution of surface ion pairs is of the order of 10 kcal/mol (Matthew, 1985), and these pairs are less likely to be conserved than buried pairs (Kumar and Nussinov, 2002). The contributions of buried ion pairs to overall stability vary depending on their geometries and the extent of burial, but can be quite high (~5 kcal/mol or higher) (Honig et al., 1986; Kumar and Nussinov, 2002). Complete ion pairs, where there are two interactions between the same two residues, are more numerous and stronger than so-called “incomplete” ion pairs, where one residue has interactions with two different residues (Gowri Shankar et al., 2007). This could allow for local flexibility. However, incomplete ion pairs are able to

form networks, and are therefore likely to contribute to the stability of the protein. Ion pairs contribute to the stability of secondary structures such as α-helices and

β-turns, but are more important for anchoring tertiary structure than secondary

structure (Gowri Shankar et al., 2007).

The fact that the species making up salt bridges are titratable means that ion pair interactions are pH-dependent. Addition of protons to the solution will change the ionisation status of the charged groups according to their pKa values, which may

be shifted in the protein environment from that of model compounds. The pKa

values of Lys and Arg are high, so these residues are protonated at physiological pH. The pKa values for the carboxyl groups on Asp and Glu are around 4, so they

are negatively charged at physiological pH. His, with a pKa value around 6.1 for

the imidazole group, is a critical potential “pH sensor” within proteins, since it will be neutralised below its pKa. Deviations in pKa values of charged residues of

up to 2 to 3 units have been observed in some proteins compared to model compounds, shifts that are attributed to electrostatic interactions and the local dielectric environment of the charged species (Matthew, 1985). Amino acids with anomalously shifted pKa values contribute significantly to the electrostatic

component of stability (Pace et al., 1990).

The conformational state of a protein which has the higher affinity for protons will be the state stabilised with a drop in pH (Pace, 1990; Whitten and García-Moreno, 2000). Thus if the acidic residues in the native state have a higher affinity for protons than those in the unfolded state, the native state will be stabilised with a decrease in pH, and vice versa. Much research has been done on the effect of pH on proteins, with the unsurprising conclusion that pH has a profound effect on the structure and stability of proteins. Additionally, in many cases, an acid-induced intermediate state is found to be stabilised by a drop in pH (Jiang et al., 1991; Muga et al., 1993; Fink et al., 1994; Whitten and García-Moreno, 2000; Chenal et

Van der Waals forces are much weaker than ionic interactions, and arise from electrostatic interactions between molecules with permanent and/or induced dipoles. The carbonyl and amide groups of the peptide backbone have permanent dipole moments, an important fact for α-helices, since the arrangement of the backbone in the helix results in a macrodipole, positive toward the N-terminus and negative toward the C-terminus (Voet and Voet, 1995). Thus, despite the relative weakness of these interactions, which attenuate with r-3, their number and arrangement within the low dielectric constant interior of a protein, means that they are a significant force in protein stability. Transient dipole moments can arise in non-polar groups due to fluctuating electron motion. Interactions between these transient dipoles are termed London dispersion forces. These are extremely weak interactions, and attenuate with r-6, such that they are only significant between contacting atoms. Correct packing of the side-chains of the protein ensures that there are a large number of interatomic contacts, and it is this which makes these forces an important factor in maintaining stability, although they are not a dominant force in folding (Liang and Dill, 2001).