Increasing protein stability by engineering the n → π* interaction at the β-turn.
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ABSTRACT:
SUBMITTER: Khatri B
PROVIDER: S-EPMC8161691 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.
| S-EPMC2084202 | biostudies-literature
Project description:Chignolin, which consists of 10 amino acids, adopts two stable states in simulations at room temperature at 1 atm: the native and misfolded states. The sequence of chignolin is optimized to form a stable π-turn and thus the native state has a π-turn from Asp3 to Thr8. On the other hand, the misfolded state adopts an α-turn from Asp3 to Gly7. We previously investigated the differences in the stability mechanism of the two states using computational techniques. Our previous detailed energy analysis implied that the native state was stabilized by hydrogen bonding between the side chain atoms of Thr6 and Thr8, and Thr8 was not involved in stabilization of the misfolded state. Thus, we predicted that mutation of Thr8 to a neutral amino acid could stabilize the misfolded structure over the native structure. In the present work, we performed 4 μs molecular dynamics simulations for 19 mutants of the 8th residue. Among them, the T8I, T8F, T8P, T8N, and T8Y mutants, in which the 8th residue was changed to a neutral residue, formed only the misfolded structure at room temperature. Even at high temperature, for the T8P mutant, the native structure was not observed, as the T8P mutant cannot form the native structure because of steric hindrance caused by the distinctive cyclic structure of proline. Interestingly, the T8P mutant at high temperature has trans and cis conformations in the Gly7-Pro8 sequence, with the trans conformation corresponding to the misfolded state. NMR analysis of the T8P mutant supported our results.
| S-EPMC9054626 | biostudies-literature
Project description:Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability.
| S-EPMC2767464 | biostudies-literature
Project description:In many common protein secondary structures, such as alpha-, 3(10), and polyproline II helices, an n --> pi* interaction places the adjacent backbone amide carbonyl groups in close proximity to each other. This interaction, which is reminiscent of the Burgi-Dunitz trajectory, involves delocalization of the lone pairs (n) of the oxygen (O(i-1)) of a peptide bond over the antibonding orbital (pi*) of C(i)=O(i) of the subsequent peptide bond. Such a proximal arrangement of the amide carbonyl groups should be opposed by the Pauli repulsion between the lone pairs (n) of O(i-1) and the bonding orbital (pi) of C(i)=O(i). We explored the conformational effects of this Pauli repulsion by employing common peptidomimetics, wherein the n --> pi* interaction is attenuated while the Pauli repulsion is retained. Our results indicate that this Pauli repulsion prevents the attainment of such proximal arrangement of the carbonyl groups in the absence of the n --> pi* interaction. This finding indicates that the poor mimicry of the amide bond by many peptidomimetics stems from their inability to partake in the n --> pi* interaction and emphasizes the quantum-mechanical nature of the interaction between adjacent amide carbonyl groups in proteins.
| S-EPMC2874455 | biostudies-literature
Project description:BACKGROUND: Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. RESULTS: Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3-4 degrees C higher over wild type. All mutants showed 2-fold higher thermal stability at 55 degrees C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. CONCLUSIONS: We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity.
| S-EPMC551599 | biostudies-literature
Project description:A series of probes with a turn-on switch for the p53-MDM2 protein-protein interaction were developed. After careful evaluation, these small molecule fluorescent probes exhibited high practical activity and selectivity in vitro and in cellulo. In particular probe 10, which had a Ki value of 0.03 ?M, displayed much better binding affinity compared to the positive control Nutlin-3, which had a Ki value of 0.23 ?M. These no-wash environment-sensitive turn-on fluorescent probes have been successfully applied to imaging p53-MDM2 interaction in the human lung cancer cell line A549 (wild-type p53) at the micromolar level. Therefore, these fluorescent probes are expected to be used in drug screening and cell staining in p53-MDM2 fields, as well as in pathological and physiological studies of the p53-MDM2 interaction.
| S-EPMC6072338 | biostudies-literature
Project description:β-Turns are secondary structure elements not only exposed on protein surfaces, but also frequently found to be buried in protein-protein interfaces. Protein engineering so far considered mainly the backbone-constraining properties of synthetic β-turn mimics as parts of surface-exposed loops. A β-turn mimic, Hot═Tap, that is available in gram amounts, provides two hydroxyl groups that enhance its turn-inducing properties besides being able to form side-chain-like interactions. NMR studies on cyclic hexapeptides harboring the Hot═Tap dipeptide proved its strong β-turn-inducing capability. Crystallographic analyses of the trimeric fibritin-foldon/Hot═Tap hybrid reveal at atomic resolution how Hot═Tap replaces a βI'-turn by a βII'-type structure. Furthermore, Hot═Tap adapts to the complex protein environment by participating in several direct and water-bridged interactions across the foldon trimer interface. As building blocks, β-turn mimics capable of both backbone and side-chain mimicry may simplify the design of synthetic proteins.
| S-EPMC2972999 | biostudies-literature
Project description:We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.
| S-EPMC2280049 | biostudies-literature
Project description:Carbonyl-carbonyl interactions between adjacent backbone amides have been implicated in the conformational stability of proteins. By combining experimental and computational approaches, we show that relevant amidic carbonyl groups associate through an n→π* donor-acceptor interaction with an energy of at least 0.27 kcal/mol. The n→π* interaction between two thioamides is 3-fold stronger than between two oxoamides due to increased overlap and reduced energy difference between the donor and acceptor orbitals. This result suggests that backbone thioamide incorporation could stabilize protein structures. Finally, we demonstrate that intimate carbonyl interactions are described more completely as donor-acceptor orbital interactions rather than dipole-dipole interactions.
| S-EPMC3742804 | biostudies-literature
Project description:[structure: see text] The trans/cis ratio of the amide bond in N-formylproline phenylesters correlates with electron withdrawal by a para substituent. The slope of the Hammett plot (rho = 0.26) is indicative of a substantial effect. This effect arises from a favorable n --> pi interaction between the amide oxygen and ester carbonyl. In a polypeptide chain, an analogous interaction can stabilize the conformation of trans peptide bonds, alpha-helices, and polyproline type-II helices.
| S-EPMC2625295 | biostudies-literature