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Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.

Lee CW, Wang HJ, Hwang JK, Tseng CP - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Systems Biology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.

ABSTRACT
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.

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Flowchart of salt-bridge design in this study.Identification of potential positions and mutation types for a given protein structure are demonstrated.
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pone-0112751-g003: Flowchart of salt-bridge design in this study.Identification of potential positions and mutation types for a given protein structure are demonstrated.

Mentions: We further classified the surface salt bridges based on the secondary structure locations. Figure 2 shows the frequency distributions of K–D, K–E, R–D, and R–E salt-bridges corresponding to different secondary structure pairs. The highest frequency occurred at the helix-helix structure for all pairings of the salt bridges except the R–D pairings. Thus, such salt bridge formation at the helix-helix structure may efficiently stabilize protein structures. Based on previous studies, the formation of R–E salt-bridge at the helix−helix structure may account for much of the stabilization of certain thermophilic proteins [41]. To determine the exact value standing for the preference of salt bridge formation of a particular type of ion pair, we constructed a matrix of empirical weights () based on the frequency of each type of salt bridges. To calculate the frequency of each type of salt bridges, the 10,556 surface salt-bridge pairs were first classified based on pairing residues, Cα–Cα distances, and secondary structure locations. Table S1 shows the empirical weight of each salt bridge type depending on the frequency. The weight matrix was then used in the weighted electrostatic attraction model to score each pairings. Finally, the angular constraints and the weighted electrostatic attraction model could be used in the last steps of identifying the pairings and the substitutions after the selection of potential positions for salt-bridge design (Figure 3).


Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.

Lee CW, Wang HJ, Hwang JK, Tseng CP - PLoS ONE (2014)

Flowchart of salt-bridge design in this study.Identification of potential positions and mutation types for a given protein structure are demonstrated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231051&req=5

pone-0112751-g003: Flowchart of salt-bridge design in this study.Identification of potential positions and mutation types for a given protein structure are demonstrated.
Mentions: We further classified the surface salt bridges based on the secondary structure locations. Figure 2 shows the frequency distributions of K–D, K–E, R–D, and R–E salt-bridges corresponding to different secondary structure pairs. The highest frequency occurred at the helix-helix structure for all pairings of the salt bridges except the R–D pairings. Thus, such salt bridge formation at the helix-helix structure may efficiently stabilize protein structures. Based on previous studies, the formation of R–E salt-bridge at the helix−helix structure may account for much of the stabilization of certain thermophilic proteins [41]. To determine the exact value standing for the preference of salt bridge formation of a particular type of ion pair, we constructed a matrix of empirical weights () based on the frequency of each type of salt bridges. To calculate the frequency of each type of salt bridges, the 10,556 surface salt-bridge pairs were first classified based on pairing residues, Cα–Cα distances, and secondary structure locations. Table S1 shows the empirical weight of each salt bridge type depending on the frequency. The weight matrix was then used in the weighted electrostatic attraction model to score each pairings. Finally, the angular constraints and the weighted electrostatic attraction model could be used in the last steps of identifying the pairings and the substitutions after the selection of potential positions for salt-bridge design (Figure 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Systems Biology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus