About

Homme Wytzes Hellinga is the James B. Duke Distinguished Professor of Biochemistry and a Professor of Biochemistry at Duke University. He is a primary faculty member in the Duke Department of Biochemistry, working within the Hellinga Lab. His research focuses on biochemistry, and he is involved in teaching and mentoring within the department. His contact email is hwh@biochem.duke.edu, and he is based at 413D Nanaline H Duke, Research Drive, Durham, NC 27708.

Research topics

• Biology
• Computer Science
• Chemistry
• Artificial Intelligence
• Computational chemistry
• Statistical physics
• Physics
• Biological system
• Biochemistry
• Microbiology
• Pharmacology

Selected publications

• Accurate Identification of Periplasmic Urea-binding Proteins by Structure- and Genome Context-assisted Functional Analysis

  Journal of Molecular Biology · 2024-09-04 · 1 citations

  articleOpen accessSenior author

  ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here.

  PublisherDOI

• Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics

  Nucleic Acids Research · 2023-05-11 · 8 citations

  articleOpen access

  DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

  PublisherOA PDFDOI

• Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching

  Communications Chemistry · 2023-08-19 · 2 citations

  articleOpen accessSenior author

  Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent response mechanisms which can be challenging to establish. We installed thiol-reactive derivatives of the naphthalene-based fluorophore Prodan into bacterial periplasmic glucose-binding proteins. Glucose binding elicited paired color exchanges in the excited and ground states of these conjugates. X-ray structures and mutagenesis studies established that glucose-mediated color switching arises from steric interactions that couple protein conformational changes to twisting of the Prodan carbonyl relative to its naphthalene plane. Mutations of residues contacting the carbonyl can optimize color switching by altering fluorophore conformational equilibria in the apo and glucose-bound proteins. A commonly accepted view is that Prodan derivatives report on protein conformations via solvatochromic effects due to changes in the dielectric of their local environment. Here we show that instead Prodan carbonyl twisting controls color switching. These insights enable structure-based biosensor design by coupling ligand-mediated protein conformational changes to internal chromophore twists through specific steric interactions between fluorophore and protein.

  PublisherOA PDFDOI

• Structure-Guided Discovery of Potent Antifungals that Prevent Ras Signaling by Inhibiting Protein Farnesyltransferase

  Journal of Medicinal Chemistry · 2022 · 12 citations

  • Chemistry
  • Biochemistry
  • Pharmacology

  levels of enzyme and farnesyl substrate. We elucidated how chemical modifications of the antifungals encode desired inhibitor conformation and concomitant inhibitory mechanism.

  PublisherOA PDFDOI

• Discovery of Thermostable, Fluorescently Responsive Glucose Biosensors by Structure-Assisted Function Extrapolation

  Biochemistry · 2022-01-27 · 4 citations

  articleSenior authorCorresponding

  Accurate assignment of protein function from sequence remains a fascinating and difficult challenge. The periplasmic-binding protein (PBP) superfamily present an interesting case of function prediction because they are both ubiquitous in prokaryotes and tend to diversify through gene duplication “explosions” that can lead to large numbers of paralogs in a genome. An engineered version of the moderately thermostable glucose-binding PBP from Escherichia coli has been used successfully as a reagentless fluorescent biosensor both in vitro and in vivo. To develop more robust sensors that meet the challenges of real-world applications, we report the discovery of thermostable homologues that retain a glucose-mediated conformationally coupled fluorescence response. Accurately identifying a glucose-binding PBP homologue among closely related paralogs is challenging. We demonstrate that a structure-based method that filters sequences by residues that bind glucose in an archetype structure is highly effective. Using fully sequenced bacterial genomes, we found that this filter reduced high paralog numbers to single hits in a genome, consistent with the accurate separation of glucose binding from other functions. We expressed engineered proteins for eight homologues, chosen to represent different degrees of sequence identity, and tested their glucose-mediated fluorescence responses. We accurately predicted the presence of glucose binding down to 31% sequence identity. We have also successfully identified suitable candidates for next-generation robust, fluorescent glucose sensors.

  PublisherDOI

• Describing Complex Structure-Function Relationships in Biomolecules at Equilibrium

  Journal of Molecular Biology · 2020 · 8 citations

  Senior authorCorresponding
  • Computer Science
  • Artificial Intelligence
  • Chemistry
  PublisherDOI

• Harnessing Environmental Ca²⁺ for Extracellular Protein Thermostabilization

  Biochemistry · 2020-09-11 · 2 citations

  articleSenior authorCorresponding

  Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose–galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

  PublisherDOI

• Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

  Proceedings of the National Academy of Sciences · 2017-05-22 · 49 citations

  articleOpen access

  Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

  PublisherOA PDFDOI

• A Unified Picture of Nucleotide Selection by a High Fidelity DNA Polymerase I

  The FASEB Journal · 2015-04-01

  articleSenior author

  Structural studies on a high-fidelity Bacillus DNA polymerase I large fragment (Bacillus fragment, BF) on deoxyribonucleotide selectivity over dideoxy- and ribo-nucleotide prior to chemistry revealed several intermediate conformations that trap incorrect substrates along the trajectory for fingers domain closure and the substrates are misaligned at the active site for incorporation [1]. Here we present structures of base-pair mismatches G•G (primer•template), T•G, and T•T bound at the BF DNA polymerase active site prior to chemistry. Comparisons of the structures of mismatches with those of cognate base pairs show that mismatches with non-cognate base pairing schemes are trapped at intermediate polymerase conformations and their functional groups are misaligned for chemistry. Among them, an extreme is the G•G mismatch which adopts a novel nucleotide binding mode that is drastically different from the previously observed ones illustrating the dynamic nature of nucleotide binding and trapping process. These structures were further analyzed in comparison with previously determined high-resolution structures of BF polymerase (more than 60) with cognate base pairs [1-5], mismatches [5-7], lesions [4, 8-10], and incorrect sugar substrates [1] bound at different fidelity filter sites. A unified picture of nucleotide selection by DNA polymerase will be presented.

  PublisherDOI

• Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation

  Journal of Neuroscience · 2013-10-09 · 119 citations

  articleOpen access

  We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R29GM049871

  NIH · $534k · 1999

• NIH Grant R01GM049871

  NIH · $2.3M · 2008

• NIH Grant DP1OD000122

  NIH · $3.8M · 2010

Frequent coauthors

• L.S. Beese

  Duke University Hospital

  57 shared

• John P. Caradonna

  Boston University

  43 shared

• Malin Allert

  Duke Medical Center

  38 shared

• Loren L. Looger

  36 shared

• M.J. Cuneo

  St. Jude Children's Research Hospital

  27 shared

• Wei Yang

  Hunan University of Science and Engineering

  27 shared

• Jenny J. Yang

  Georgia State University

  27 shared

• Yiming Ye

  Key Laboratory of Guangdong Province

  25 shared

Education

• Ph.D., Biochemistry

  University of Groningen

  1976

• M.S., Biochemistry

  University of Groningen

  1973

• B.S., Biochemistry

  University of Groningen

  1971