Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a Gly-to-Ser mutation

Berndt, Kurt D.; Beunink, J; Schröder, Werner; Wüthrich, Kurt

doi:10.1021/bi00068a012

Cited by 58 publications

(65 citation statements)

References 33 publications

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“…We are aware of three cases in which this has been demonstrated experimentally. The first two are the G36S mutant in basic pancreatic trypsin inhibitor (Berndt et al 1993) and the N52I mutant in Iso-1-cyto- a Mutant Q105E was constructed in the WT rather than the WT* background (Pjura et al 1993). chrome c (Lett et al 1996). The third example is provided by the binding pocket of sperm whale deoxymyoglobin that contains an internal water molecule that forms a single hydrogen bond to His-64.…”

Section: Resultsmentioning

confidence: 99%

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

Baase

Quillin

et al. 2001

Protein Science

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To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7Å, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solventbinding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

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Section: Resultsmentioning

confidence: 99%

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

Baase

Quillin

et al. 2001

Protein Science

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“…The assertion that one of the four internalwatermoleculesofBPTIexchangestooslowly to contribute to the 17 O dispersion is supported by the 2 H relaxation data presented in the accompanying paper (Denisov & Halle, 1995). A decisive test of these tentative assignments would be to measure the 17 O relaxation dispersion from BPTI mutants with one or more of the four internal water molecules displaced (Housset et al, 1991;Berndt et al, 1993).…”

Section: Internal Watermentioning

confidence: 91%

Protein Hydration Dynamics in Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion

Денисов

Halle

1995

Journal of Molecular Biology

162

177

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Water oxygen-17 spin relaxation was used to study hydration and dynamics Condensed Matter Magnetic Resonance Group, Chemical of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal Center, Lund University P. O. Box 124, S-22100 Lund and transverse relaxation rates was measured over the Larmor frequency Sweden range 2.6 to 49 MHz in the pD range 2 to 11 at 27°C. While the protein-induced relaxation enhancement was similar for the two proteins at high frequencies, it was an order of magnitude smaller for ubiquitin than for BPTI at low frequencies. This difference was ascribed to the abscence, in ubiquitin, of highly ordered internal water molecules, which are known to be present in BPTI and in most other globular proteins. These observations demonstrate that the water relaxation dispersion in protein solutions is essentially due to a few structural water molecules buried within the protein matrix, but exchanging rapidly with the external water. The relaxation data indicate that the internal water molecules of BPTI exchange with bulk water on the time-scale 10 −8 to 10 −6 second thus lowering the recently reported upper bound on the residence time of these internal water molecules by four orders of magnitude, and implying that local unfolding occurs on the submicrosecond time-scale. The water molecules residing at the surface of the two proteins were found to be highly mobile, with an average rotational correlation time of approximately 20 picoseconds. For both proteins, the oxygen-17 relaxation depended only very weakly on pD, showing that ionic residues do not perturb hydration water dynamics more than other surface residues. We believe that the present results resolve the long-standing controversy regarding the mechanism behind the spin relaxation dispersion of water nuclei in protein solutions, thus establishing oxygen-17 relaxation as a powerful tool for studies of structurally and functionally important water molecules in proteins and other biomolecules.

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“…In referring to both isozymes in the text, we use the numbering given in Table 1. that Ser-37 is also in a hydrophobic environment because the sequence surrounding Ser-37 in the isozyme studied by NMR contains only uncharged or hydrophobic residues. In dendrotoxin K, a slowly exchanging serine residue (Ser-36) is also found in a loop and this exchange behavior has been ascribed to multiple hydrogen bond interactions (Berndt et al, 1993) The crystal structure of 4-OT shows that the imidazole side chain of His-49, a residue located in the second turn near the active site, is solvent exposed. This is surprising because a pK, of 5.2 for this residue was measured by proton NMR spectroscopy (Stivers et al, 1996a).…”

Section: Comparison Of the Nmr And Crystal Structurementioning

confidence: 99%

4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

Stivers¹,

Abeygunawardana²,

Whitman³

et al. 1996

Protein Science

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Abstract4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated a-keto acids using Pro-1 as a general base (Stivers et al., 1996a(Stivers et al., , 1996b. We report the backbone and side-chain 'H, "N, and I3C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an a-helix (residues 13-30), two 0-strands (PI, residues 2-8; &, residues 39-45), a @-hairpin (residues 50-57), two loops (I, residues 9-12; 11, 34-38), and two turns (I, residues 30-33; II,47-50). The remaining residues form coils. The PI strand is parallel to the pz strand of the same subunit on the basis of cross strand NHi-NHj NOEs in a 2D "N-edited 'H-NOESY spectrum of hexameric 4-OT containing two I5N-labeled subunits/hexamer. The 0' strand is also antiparallel to another PI strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from I5N T l / T , measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-0~0-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide I5N and NH chemical shifts of residues in the 0-hairpin and in loop 11, providing evidence that these regions change conformation when the active site is occupied.

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Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a Gly-to-Ser mutation

Cited by 58 publications

References 33 publications

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

Protein Hydration Dynamics in Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion

4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

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