Searching the Resource Information Network
Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.
Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.
Pubmed ID: 22279578
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
A searchable database of pKa values of amino acid residues within proteins. These values have been measured experimentally using a variety of methods, including NMR and the linearized Poisson-Boltzmann equation. This database of protein ionization constants was sourced from the primary literature and contains in excess of 1400 entries. The database contains pKa values for amino acid side-chains, as well as the N and C termini, over 75% of which focus on Glutamate, Lysine, Histidine and Aspartate. These four residues are all key ionizable residues, and therefore the apparent bias is not driven by our selection, but by the available experimental data. Very little data is currently available for Arginine: its pKa value (~12) essentially precludes measurement by titration as proteins will denature at high basic pH.
View all literature mentionsDelPhi provides numerical solutions to the Poisson-Boltzmann equation (both linear and nonlinear form) for molecules of arbitrary shape and charge distribution. The current version is fast, accurate, and can handle extremely high lattice dimensions. It also includes flexible features for assigning different dielectric constants to different regions of space and treating systems containing mixed salt solutions. DelPhi takes as input a coordinate file format of a molecule or equivalent data for geometrical objects and/or charge distributions and calculates the electrostatic potential in and around the system, using a finite difference solution to the Poisson-Boltzmann equation. DelPhi is a versatile electrostatics simulation program that can be used to investigate electrostatic fields in a variety of molecular systems. Features of DelPhi include solutions to mixtures of salts of different valence; solutions to different dielectric constants to different regions of space; and estimation of the best relaxation parameter at run time.
View all literature mentions