Biology subjects

Pyles, H.

Publications and source records attributed to Pyles, H..

5 recordsLinked to original sources

bioRxiv2025

Inhibition of ice recrystallization with designed twistless helical repeat proteins

Given the repetitive structure of crystalline ice, its unsurprising that highly active ice-binding proteins, often with beta-roll structures, also have repeating motifs. Here, we introduce a de novo designed family of ice-binding twistless alpha-helical repeat (iTHR) proteins. Each iTHR protein comprises two planar layers of parallel alpha-helices connected by loops--a structural topology not seen in native ice-binding proteins. The ice-binding helices feature an ordered array of TXXXAXXXAXX motifs, spaced to match the pyramidal {201} and secondary prism plane {110} ice lattice facets, and engineered with a 98.2{degrees} turn angle per residue to orient threonines uniformly towards the ice surface. iTHR proteins show high solubility, thermostability, and produce varied ice crystal morphologies depending on their intended target facet. Crucially, iTHRs exhibit ice recrystallization inhibition (IRI) at critical concentrations comparable to those of many native globular ice-binding proteins. Extensive site-specific mutagenesis shows that ice-binding activity in iTHR proteins is robust, remaining largely unaffected by changes in chemical composition. Variation in the repeat number reveals a non-monotonic relationship to IRI activity. X-ray crystal structures of two designs confirm the intended orientation of threonines, uniformly pointing toward the ice surface. The iTHR family provides a versatile platform to systematically investigate the complex structure-activity relationships underlying protein-ice interactions.

biophysics↗

bioRxiv2024

Protein identification using cryo-EM and artificial intelligence guides improved sample purification

Protein purification is essential in protein biochemistry, structural biology, and protein design. It enables the determination of protein structures, the study of biological mechanisms, and the biochemical and biophysical characterization of both natural and de novo designed proteins. Despite the broad application of various protein purification protocols, standard strategies can still encounter challenges, such as the unintended co-purification of unknown contaminants alongside the target protein. In particular, co-purification issues pose significant challenges for designed self-assembling protein nanomaterials, as it is difficult to determine whether unexpected observed geometries represent novel assembly states of the designed system, cross-contamination from other assemblies, or native proteins originating from the expression host. In this study, we assessed the ability of an automated structure-to-sequence pipeline to unambiguously identify an unknown co-purifying protein found across several purified designed protein samples. Using cryo-electron microscopy (Cryo-EM), ModelAngelos sequence-agnostic automated model-building feature, and the Basic Local Alignment Search Tool (BLAST), we identified the unknown protein as dihydrolipoamide succinyltransferase (DLST). This identification was further confirmed by comparing the cryo-EM data with available DLST structures in the Protein Data Bank (PDB) and AlphaFold 3 predictions from the top BLAST hits. The clear identification of DLST informed our subsequent literature search and led to the rational modification of our protein purification protocol, ultimately enabling the exclusion of the contaminant from preparations of our target nanoparticle. This study demonstrates the successful application of a structure-to-sequence workflow, integrating Cryo-EM, ModelAngelo, protein BLAST, PDB structures, and AlphaFold 3 predictions, to identify and remove an unknown protein from a purified sample. It also highlights the broader potential of integrating Cryo-EM with AI-driven tools for accurate protein identification across various samples and contexts within protein science. HighlightsO_LIAn unknown protein was consistently found in multiple de novo designed protein samples. C_LIO_LIThe protein was identified as dihydrolipoamide succinyltransferase (DLST) using Cryo-EM, ModelAngelo and BLAST, and further verified using AlphaFold 3 and the PDB. C_LIO_LIIdentification enabled rational modification of the purification protocol to exclude the contaminant. C_LIO_LIThis method enables accurate protein identification without requiring near-atomic resolution or prior sequence and structural data, making it broadly applicable to various areas of protein science. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/612515v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@592410org.highwire.dtl.DTLVardef@4ea3f3org.highwire.dtl.DTLVardef@edd0bcorg.highwire.dtl.DTLVardef@122fba2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Controlling semiconductor growth with structured de novo protein interfaces

Protein design now enables the precise arrangement of atoms on the nanometer length scales of inorganic crystal nuclei, opening up the possibility of templating the growth of metal oxides including semiconductors. We designed proteins presenting regularly repeating interfaces containing functional groups that organize ions and water molecules, and characterized their ability to bind to and template metal oxides. Two interfaces promoted the growth of hematite under conditions that otherwise resulted in the formation of magnetite. Three interfaces promoted ZnO nucleation under conditions where traditional ZnO-binding peptides and control proteins were ineffective. Designed cyclic assemblies with these ZnO nucleating interfaces lining interior cavities promoted ZnO growth within the cavity. CryoEM analysis of a designed octahedral nanocage revealed atomic density likely corresponding to the growing ZnO directly adjacent to the designed nucleation promoting interfaces. These findings demonstrate that designed proteins can direct the formation of metal oxides not observed in biological systems, opening the door to protein-semiconductor hybrid materials. One Sentence SummaryWe describe the design of structured protein interfaces that bind to, promote, and localize the growth of zinc oxide and hematite, inorganic materials which are not found in biological systems.

bioengineering↗

bioRxiv2023

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

biochemistry↗

bioRxiv2023

Directing polymorph specific calcium carbonate formation with de novo protein templates

Biomolecules modulate inorganic crystallization to generate hierarchically structured biominerals1-5, but the atomic structure of the organic-inorganic interfaces that regulate mineralization remain unknown6-8. We hypothesized that heterogeneous nucleation of calcium carbonate could be achieved by a structured flat molecular template that pre-organizes calcium ions on its surface. To test this hypothesis, we designed helical repeat proteins (DHRs) displaying regularly spaced carboxylate arrays on their surfaces and found that both protein monomers and protein-Ca2+ assemblies directly nucleate nano-calcite with non-natural (110) or (202) faces while vaterite, which forms first absent the proteins, is bypassed. The nanocrystals then assemble by oriented attachment into calcite mesocrystals. We find further that nanocrystal size and polymorph can be tuned by varying the length and surface chemistry of the designed protein templates. Thus, bio-mineralization can be programmed using de novo protein design, providing a route to next-generation hybrid materials. One sentence summaryDe novo designed protein templates promote nucleation of nano-calcite and direct its growth by oriented particle attachment.

biochemistry↗