PDBsum entry 1tt2

Hydrolase

PDB id

1tt2

Contents

			Protein chain

					130 a.a. *

			Ligands

					DMS ×2


					THP


					GOL

			Metals

					_CA

			Waters ×116

* Residue conservation analysis

References listed in PDB file

Key reference

Title

X-Ray and thermodynamic studies of staphylococcal nuclease variants i92e and i92k: insights into polarity of the protein interior.

Authors

D.M.Nguyen, R.Leila reynald, A.G.Gittis, E.E.Lattman.

Ref.

J Mol Biol, 2004, 341, 565-574. [DOI no: 10.1016/j.jmb.2004.05.066]

PubMed id

15276844

Abstract

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.



	Figure 3. Figure 3. Ribbon representation of the superposition of the I92E cryo (cyan) and (a) I92E room temperature (red) structures and (b) the I92K cryo structure. (a) The side- chain of 92E in both cryo (blue) and room temperature (orange) adopts an identical conformation. The blue spheres represent the four completely buried and the two bridging water molecules seen in the I92E cryo structure. (b) The two conformations of 92K as observed in the I92K cryo structure with hydrophobic residues that surround conformers 1 (magenta) and conformer 2 (green) within 4 Å .		Figure 5. Figure 5. The two conformations of 92K built into the 2Fo 2 Fc (blue) and Fo 2 Fc (magenta) electron density maps, contoured at 1s and 3.5s respectively, calculated from a model that had alanine at position 92. The sub- sequent refinement of the model with the alternate 92K conformations showed no electron density for the C d and N z atoms for conformer 1 or for the N z for conformer 2, which is indicative of increased mobility of the 92K side-chain. The electron density maps displayed might suggest the native isoleucine at this position. The lysine mutation was confirmed by DNA sequencing and mass spectrometry (data not shown). Moreover, none of the four most frequent isoleucine conformers could be built into these electron density maps (see Supplementary Material).

PROCHECK

	Headers