Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

Pakula, Andrew A.; Sauer, Robert T.

doi:10.1038/344363a0

Cited by 159 publications

(114 citation statements)

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“…Other factors, such as the efficiency of hydrophobic packing in the protein core (e.g., see Lim & Sauer, 1991, and references therein), reverse hydrophobic effects of protein surface residues (Pakula & Sauer, 1990), or electrostatic stabilization due to the nonconservatively substituted Lys 6 within the metal-binding loop may also contribute to the enhanced thermal stability of P. furiosus Rd. NMR studies of the temperature-dependent structural and dynamic properties of P. furiosus Rd are now underway.…”

Section: Discussionmentioning

confidence: 99%

Solution‐state structure by NMR of zinc‐substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

et al. 1992

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The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 " c . Structures were generated with DSPACEB by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (N0E)-derived interproton distance restraints, including 67 interresidue, 124 sequential ( i -j = I), 75 medium-range ( i -j = 2-5), and 137 long-range ( i -j > 5) restraints.All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backboneside chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to >16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridiumpusteuriunum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +standard deviation) of 0.024 f 0.003 A 2 and a maximum total penalty of 0.035 A*. Superposition of the backbone atoms (C, Ca, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (fSD) of 0.42 f 0.07 A. Superposition of all heavy atoms for residues AI-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 k 0.08 A . Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pusteuriunum, with the average rms deviation value for backbone atoms of residues Al-LSI of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pusteuriunum Rd of 0.77 +_ 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions. Four of these interactions involve the nonconservatively substituted Glu 14, Ala 1, and Trp 3 residues. The combined findings are consistent with the proposal that stabilization of the N-terminal residues inhibits the &sheet from "unzipping" at elevated temperatures (Blake, P.R., Park, J.-B., Bryant, EO., et al., 1991, Biochemistry30, 10885-10895). In view of the high structural similarities between this hyperthermophilic protein and C...

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Section: Discussionmentioning

confidence: 99%

Solution‐state structure by NMR of zinc‐substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

et al. 1992

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“…A variety of successful routes to this objective has been undertaken. These include: (1) introducing disulfide bridges (Matsumura et al, 1989b); (2) increasing internal hydrophobic packing (Malcolm et al, 1990); (3) reducing solvent-exposed nonpolar surface area (Pakula & Sauer, 1990); (4) incorporating a metal association site (Kuroki et al, 1989); and (5) adding charged side chains to interact with the a-helix dipoles (Nicholson et al, 1988). However, the above strategies are not always successful and their pursuit has often yielded proteins that are less stable than the WT progenitors for reasons that are not fully understood (e.g., Karpusas et al, 1989;Matsumura et al, 1989a;Eijsink et al, 1992;Leontiev et al, 1993 Abbreviations: hs, a hyperstable variant of chicken lysozyme; GdnHCI, guanidine hydrochloride; T,, the midpoint temperature of the thermal denaturation transition; C,, the midpoint concentration of the GdnHCl denaturation profile; DSC, differential scanning calorimetry; AH,,,, calorimetrically determined enthalpy of denaturation; AC,, the difference between the heat capacities of the native and the denatured states; WT or wt, wild type.…”

Section: Introductionmentioning

confidence: 99%

Design and structural analysis of an engineered thermostable chicken lysozyme

Shih

Kirsch

1995

Protein Science

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A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (AT, = + 10.5 "C) and chemical (AC,n for guanidine hydrochloride denaturation = + 1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 "C) to 4.0 (differential scanning calorimetry, 74 "C) kcal mol" greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The 155L/S91T core construct with AT, = 3.3 "C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062. (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 3 1 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The DlOlS variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.Keywords: a-helix propensity; avian lysozyme sequences; chicken lysozyme; human lysozyme; interior packing; protein design; structures; thermodynamic stability A challenging goal of protein engineering is the design of proteins with significantly enhanced thermo-and chemical stabilities. A variety of successful routes to this objective has been undertaken. These include: (1) introducing disulfide bridges (Matsumura et al., 1989b); (2) increasing internal hydrophobic packing (Malcolm et al., 1990); (3) reducing solvent-exposed nonpolar surface area (Pakula & Sauer, 1990); (4) incorporating a metal association site (Kuroki et al., 1989); and (5) adding charged side chains to interact with the a-helix dipoles (Nicholson et al., 1988). However, the above strategies are not always successful and their pursuit has often yielded proteins that are less stable than the WT progenitors for reasons that are not fully understood (e.g., Karpusas et al., 1989;Matsumura et al., 1989a;Eijsink et al., 1992;Leontiev et al., 1993 Abbreviations: hs, a hyperstable variant of chicken lysozyme; GdnHCI, guanidine hydrochloride; T,, the midpoint temperature of the thermal denaturation transition; C,, the midpoint concentration of the GdnHCl denaturation profile; DSC, differential scanning calorimetry; AH,,,, calorimetrically determined enthalpy of denaturation; AC,, the difference between the heat capacities of the native and the denatured states; WT or wt, wild type.

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“…Thus, capping is independent of helix length (i.e., charge separation in the macrodipole). Tidor (1994) also found that electrostatic contributions from the macrodipole decrease markedly beyond the first/last turn, based, in this case, on simulation of a well-characterized mutation in A Cro (Pakula & Sauer, 1990). By calculating contributions to the free energy from an Ncap mutation (viz., YZ6 + D in the helix that spans residues 26-36), he concluded that the capping effect is due both to specific hydrogen bonding and to electrostatic interactions with spatially proximate groups, some nearby in sequence, some quite distant.…”

Section: Capping Studies In Simulationsmentioning

confidence: 99%

Helix capping

1998

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Helix-capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices in both proteins and peptides. In an a-helix, the first four >N-H groups and last four >C=O groups necessarily lack intrahelical hydrogen bonds. Instead, such groups are often capped by alternative hydrogen bond partners. This review enlarges our earlier hypothesis (Presta LG, Rose GD. 1988. Helix signals in proteins. Science 240:1632-1641) to include hydrophobic capping. A hydrophobic interaction that straddles the helix terminus is always associated with hydrogen-bonded capping. From a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix N-terminus and four at the C-terminus. The consensus sequence patterns of these seven motifs, together with results from simple molecular modeling, are used to formulate useful rules of thumb for helix termination. Finally, we examine the role of helix capping as a bridge linking the conformation of secondary structure to supersecondary structure.Keywords: alpha helix; protein folding; protein secondary structure The a-helix is characterized by consecutive, main-chain, i + i -4 hydrogen bonds between each amide hydrogen and a carbonyl oxygen from the adjacent helical turn (Pauling & Corey, 1951). This pattern (Fig. 1) is interrupted at helix termini because, upon termination, no turn of helix follows to provide additional hydrogen bond partners. Such end effects are substantial, encompassing two-thirds of the residues for the protein helix of average length (Presta & Rose, 1988). Further, helix geometry hinders solvent access to amide groups in the first turn of the helix, inhibiting interaction with water and necessitating alternative hydrogen bonds. The term helix "capping" (Richardson & Richardson, 1988a) has been used to describe such alternative hydrogen bond patterns that can satisfy backbone >N -H and >C = 0 groups in the initial and final turns of the helix (Presta & Rose, 1988).Many studies involving helix capping have been conducted since publication of our initial hypothesis nine years ago (Presta & Rose, 1988). As we had proposed, amide hydrogens at the helix N-terminus are indeed satisfied predominantly by side-chain H-bond acceptors. In contrast, carbonyl oxygens at the C-terminus are satisfied primarily by backbone >N -H groups from the turn following the helix. Further, these hydrogen-bonding patterns at either helix end are accompanied by a companion hydrophobic interaction between apolar residues in the a-helix and its flanking turn. This hydrophobic component of helix capping was unanticipated.The main purpose of this review is to enlarge our previous definition of helix capping and to document the common capping motifs. Qpically, protein helices terminate in a hydrophobic interaction that straddles the helix termini (i.e., an interaction between two hydrophobic residues close in sequence, one within the helix, the other external to the helix). In this interaction,...

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Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

Cited by 159 publications

References 12 publications

Solution‐state structure by NMR of zinc‐substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

Solution‐state structure by NMR of zinc‐substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

Design and structural analysis of an engineered thermostable chicken lysozyme

Helix capping

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