PDBsum entry 1eyc

Hydrolase

PDB id: 1eyc

Contents

Protein chain

135 a.a. *

Waters ×56

* Residue conservation analysis

References listed in PDB file

Key reference

• Title: Increasing the thermostability of staphylococcal nuclease: implications for the origin of protein thermostability.
• Authors: J.Chen, Z.Lu, J.Sakon, W.E.Stites.
• Ref.: J Mol Biol, 2000, 303, 125-130. [DOI no: 10.1006/jmbi.2000.4140]
• PubMed id: 11023780

Abstract

Seven hyper-stable multiple mutants have been constructed in staphylococcal nuclease by various combinations of eight different stabilizing single mutants. The stabilities of these multiple mutants determined by guanidine hydrochloride denaturation were 3.4 to 5.6 kcal/mol higher than that of the wild-type. Their thermal denaturation midpoint temperatures were 12.6 to 22.9 deg. C higher than that of the wild-type. These are among the greatest increases in protein stability and thermal denaturation midpoint temperature relative to the wild-type yet attained. There has been great interest in understanding how proteins found in thermophilic organisms are stabilized. One frequently cited theory is that the packing of hydrophobic side-chains is improved in the cores of proteins isolated from thermophiles when compared to proteins from mesophiles. The crystal structures of four single and five multiple stabilizing mutants of staphylococcal nuclease were solved to high resolution. No large overall structural change was found, with most changes localized around the sites of mutation. Rearrangements were observed in the packing of side-chains in the major hydrophobic core, although none of the mutations was in the core. It is surprising that detailed structural analysis showed that packing had improved, with the volume of the mutant protein's hydrophobic cores decreasing as protein stability increased. Further, the number of van der Waals interactions in the entire protein showed an experimentally significant increase correlated with increasing stability. These results indicate that optimization of packing follows as a natural consequence of increased protein thermostability and that good packing is not necessarily the proximate cause of high stability. Another popular theory is that thermostable proteins have more electrostatic and hydrogen bonding interactions and these are responsible for the high stabilities. The mutants here show that increased numbers of electrostatic and hydrogen bonding interactions are not obligatory for large increases in protein stability.

Figure 1.
Figure 1. Plot of protein stability (DG[H[2]O]in kcal/mol) versus the number of van der Waals interactions found in each structure. The crystals of all proteins were obtained at methyl pentane diol concentrations ranging from 40 to 60 % (v/v) in 25 mM sodium phosphate buffer of either pH 7 or 8. X-ray diffraction data were collected using an R-AXIS IV. The CuKaX-ray source was a Rigaku RU-H3RHB generator focused by Osmic-type multilayer diffraction mirrors. Data reductions were carried out using the program SCALEPACK. Manual model adjustment was performed using InsightII and refinement of positional parameters and B -factors used SHELX97. Structures have been deposited in the RCSB Protein Data Bank with the following accession numbers: wild-type 1EY0 and 1EYD, T33V 1EY5 and 1EZ8, T41I 1EY6, S59A 1EY4, S128A 1EY7, GLA 1EY8, IGLA 1EY9 VIGLA 1EYA, IAGLA 1EYC, VIAGLA 1EZ6. The criteria for interaction was to require the center to center distance for two non-bonded atoms to be equal to or less than the sum of the van der Waals radii plus 0.15 Å. This added distance was varied in 0.05 Å increments from zero to 0.3 Å. Although the absolute number of contacts varied with interaction distance cutoff, the rank order of the number of contacts found for different mutants was nearly identical at each distance and graphs similar to that above are obtained regardless of precise cutoff distance. (A Table is available as Supplementary Material that gives the number of interactions for each structure at each distance cutoff). The values plotted for wild-type and T33V contacts are the average found in three and two structures, respectively. The standard deviation of number of contacts in the three wild-type structures at this cutoff distance was ±9. However, the error bars are set at ±20 contacts, a more conservative value derived from observed variations as structure refinements progressed. The greatest standard deviation in the difference in contacts at any distance cutoff for the three wild-type structures was ±14 contacts. The expected positional uncertainty in each atom predicted from a Luzzati plot is 0.2 Å. The continuous line is the least-squares fit with R2=0.6064.

The above figure is reprinted by permission from Elsevier: J Mol Biol (2000, 303, 125-130) copyright 2000.