Protein Thermal Stability Enhancement by Designing Salt Bridges: A Combined Computational and Experimental Study

<div><p>Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα–Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K–D49, E96R–D28, E96K–D28, S440K–E70, T231K–D388, and Q277E–D282 was detected, respectively. Reversing the polarity of T231K–D388 to T231D–D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study demonstrated a novel method for the thermal adaptive design of salt bridges through inference of suitable positions and substitutions.</p></div

Relative solvent accessibility of salt bridges in 6,493 X-ray protein structures.

<p>Abbreviation: K, Lysine; R, Arginine; D, Asparagine; E, Glutamine; and RSA: Relative solvent accessibility.</p>a<p>RSA of both residues of a salt bridge are indicated.</p>b<p>RSA of one residue of a salt bridge is >25% and the other is >35%.</p><p>Relative solvent accessibility of salt bridges in 6,493 X-ray protein structures.</p

Flowchart of salt-bridge design in this study.

<p>Identification of potential positions and mutation types for a given protein structure are demonstrated.</p

Frequency distributions of surface salt bridges at different pairs of secondary structures.

<p>Four types of salt-bridge pairings, Arg/Asp (R/D), Arg/Glu (R/E), Lys/Asp (K/D), and Lys/Glu (K/E), were considered in the statistical analysis.</p

Characteristics of positions in BglA for mutations.

<p>Secondary structure (SS: H, helix; E, beta-sheet; C, coil), relative solvent accessibility (RSA), Z-scores of B-factor (z_B-factor) and reciprocal of weighted contact number (z_rWCN), conservation score, and inter- or intra- subunit location are indicated.</p><p>Characteristics of positions in BglA for mutations.</p

Locations of eight predicted pairs and one control pair in the octameric structure (PDB: 1BGA).

<p>A, D, E subunits are represented in green, yellow, and purple, respectively. The N437–D49 pair is between subunits A and D; the T231–D388 pair is between subunits A and E; whereas E96–D28, Q141–E148, Q277–R137, S440–E70 and control pair Q216-D289 are intra-subunit pairs. The predictive pairs as well as T231D–D388K and Q216R–D289 pairs were simulated by Pymol program. Each pair is magnified in an independent window showing secondary structure elements and the paired residues (red for negatively charged residues and blue for positively charged residues). The neighboring charge residue of each position (E96, Q141, T231, Q277, N437, S440, and Q216) suggested the oppositely charged substitutions for forming salt bridges.</p

Spatial orientation and Cα–Cα distances of salt bridges on protein surfaces.

<p>(A) Statitical analysis of angles (θ₁, θ₂) of 10,556 salt bridges on the surfaces of 6,493 X-ray protein structures. Two angles of a salt bridge (<i>i</i>–<i>j</i>), ∠Cβ₁Cα₁Cα₂ (θ₁) and ∠Cβ₂Cα₂Cα₁ (θ₂), were used to describe the charge group interaction based on the relative orientation of the two residues’ Cα–Cβ vectors as indicated. All of the angles are in the range of 0° to 180° (θ₁ and θ₂ color in black and gray). The length of radius are corresponding to Cα–Cα distance (Å). (B) The scatter plot shows the angles (θ₁, θ₂) of the salt bridges at a backbone distance >7 Å, which are restrained within 0° to 110°.</p