Determinants of miniprotein stability: can anything replace a buried H-bonded Trp sidechain?

A novel 20-residue fold, designated the `Trp-cage' motif, hasbeen shown to be 98+% folded in both water and 30 vol-%trifluoroethanol solution. Folding is cooperative andhydrophobically driven, resulting in the burial of the Trpsidechain and a stable H-bond from the Trp-NH to a sequenceremote backbone carbonyl. In the present study the effects ofreplacing the Trp with His, Phe and both isomers of -naphthylalanine are examined. The results suggest that thehydrophobic cluster is a specific interaction of proline ringswith the indole ring which can be partially mimicked by anaphthalene ring. The His and Phe mutants are completelyunfolded in aqueous medium. The naphthylalanine mutants forma stable hydrophobic cluster in 30% trifluoroethanol, but areless stable in water than the native structure.     

VCD spectroscopic properties of the β‐hairpin forming miniprotein CLN025 in various solvents

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the β‐hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable β‐hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a β‐hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave‐assisted solid phase peptide synthesis with N^α‐Fmoc protected amino acids. The VCD spectra displayed a (?,+,?) pattern with bands at 1640 to 1656 cm^?1, 1667 to 1687 cm^?1, and 1679 to 1686 cm^?1 formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm^?1 with component bands at 1630 cm^?1, 1646 cm^?1, 1658 cm^?1, and 1675 to 1680 cm^?1 that are indicative of the β‐sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of β‐hairpins formed by ^DPro‐Xxx turns and indicated that observed pattern is typical of β‐hairpins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 442–450, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cystine-knot peptides: emerging tools for cancer imaging and therapy

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature’s repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.     

Possible locally driven folding pathways of TC5b,a 20-residue protein

A novel computational procedure for modeling possible locally driven folding pathways by stepwise elongations of the peptide chain was successfully applied to TC5b, a 20-residue miniprotein. Systematic exploration of the possible locally driven pathways showed that the Trp-cage structure of TC5b could be obtained by stepwise elongation starting from the noncentral local nucleation centers preexisting in the unfolded state of TC5b. The probable locally driven folding pathway starts with folding of alpha-helical fragment 4-9, followed by formation of the proper three-dimensional structure of fragment 4-12, and then 4-18. Accordingly, the Trp-cage-forming interactions emerge successively, first Trp(6)-Pro(12), then Trp(6)-Pro(18), and then Trp(6)-Tyr(3). The Trp-cage-like structures of TC5b found in this study by independent energy calculations are in excellent agreement with the NMR experimental data. The same procedure rationalizes the incomplete Trp-cage formation observed for two analogs of TC5b. Generally, the success of this novel approach is encouraging and provides some justification for the use of computational simulations of locally driven protein folding.     

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.     

Integration of the Rosetta suite with the python software stack via reproducible packaging and core programming interfaces for distributed simulation

The Rosetta software suite for macromolecular modeling is a powerful computational toolbox for protein design, structure prediction, and protein structure analysis. The development of novel Rosetta‐based scientific tools requires two orthogonal skill sets: deep domain‐specific expertise in protein biochemistry and technical expertise in development, deployment, and analysis of molecular simulations. Furthermore, the computational demands of molecular simulation necessitate large scale cluster‐based or distributed solutions for nearly all scientifically relevant tasks. To reduce the technical barriers to entry for new development, we integrated Rosetta with modern, widely adopted computational infrastructure. This allows simplified deployment in large‐scale cluster and cloud computing environments, and effective reuse of common libraries for simulation execution and data analysis. To achieve this, we integrated Rosetta with the Conda package manager; this simplifies installation into existing computational environments and packaging as docker images for cloud deployment. Then, we developed programming interfaces to integrate Rosetta with the PyData stack for analysis and distributed computing, including the popular tools Jupyter, Pandas, and Dask. We demonstrate the utility of these components by generating a library of a thousand de novo disulfide‐rich miniproteins in a hybrid simulation that included cluster‐based design and interactive notebook‐based analyses. Our new tools enable users, who would otherwise not have access to the necessary computational infrastructure, to perform state‐of‐the‐art molecular simulation and design with Rosetta.     

Determinants of miniprotein stability: can anything replace a buried H-bonded Trp sidechain?

Summary A novel 20-residue fold, designated the ‘Trp-cage’ motif, has been shown to be 98+% folded in both water and 30 vol-% trifluoroethanol solution. Folding is cooperative and hydrophobically driven, resulting in the burial of the Trp sidechain and a stable H-bond from the Trp-εNH to a sequence remote backbone carbonyl. In the present study the effects of replacing the Trp with His, Phe and both isomers of β-naphthylalanine are examined. The results suggest that the hydrophobic cluster is a specific interaction of proline rings with the indole ring which can be partially mimicked by a naphthalene ring. The His and Phe mutants are completely unfolded in aqueous medium. The naphthylalanine mutants form a stable hydrophobic cluster in 30% trifluoroethanol, but are less stable in water than the native structure.     

Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease

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