Biology subjects

Ziarek, J. J.

Publications and source records attributed to Ziarek, J. J..

18 recordsLinked to original sources

bioRxiv2026

Differentiating 5-thiooxazoles from oxazolone-coupled thioamides in RiPP natural products

Conversion of cysteine residues to 5-thiooxazole moieties by multinuclear nonheme iron-dependent oxidative enzymes (MNIOs) is a prevalent modification in ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. However, this post-translational modification (PTM) is difficult to distinguish from MNIO-produced oxazolone-coupled thioamides, such as those present in the RiPP methanobactin. The RiPP virulence factor oxazolin contains six copper-binding heterocycles installed by an MNIO. Here, we reassign these PTMs, originally described as oxazolones/thioamides, as 5-thiooxazoles on the basis of detailed comparative chemical and structural characterization of oxazolin and methanobactin. These data establish a benchmark for differentiating these two PTMs in newly discovered RiPPs. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/730506v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1fc7a4eorg.highwire.dtl.DTLVardef@c1b8baorg.highwire.dtl.DTLVardef@46cf36org.highwire.dtl.DTLVardef@ce1fea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Bis-hydroxylation of Homocitrulline Catalyzed by a Multinuclear Nonheme Iron-Dependent Oxidative Enzyme during RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a Streptomyces thermodiastaticus JCM 4840 RiPP biosynthetic gene cluster, the std cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the std biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the backbone of asparagine. Next, a peptidase S8/S53 domain fused to a NodU-like carbamoyltransferase that both carbamoylates the {varepsilon}-amino group of lysine to produce the non-proteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the {beta}- and {gamma}-carbon positions of the installed homocitrulline. The formation of homocitrulline and its subsequent modification are unprecedented in RiPP biosynthesis. Moreover, these findings expand the substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyl transferases in RiPP biosynthesis.

biochemistry↗

bioRxiv2026

A bivalent lysine-acetylated small-molecule binding site in MYC

MYC is an important, yet challenging target in oncology as it lacks traditional druggable pockets. Here, we show that two regions of MYC, the basic-helix-loop-helix (bHLH) domain, and extended MYC Box II (eMBII) come together to form a bivalent, high-affinity small-molecule MYC inhibitor (MYCi) binding site. CRISPR-tiling mutagenesis identified mutations in the vicinity of the eMBII and bHLH regions that together confer MYCi resistance. Importantly, acetylation of K148 in eMBII, which is essential for MYC oncogenicity in vivo, enhanced MYCi binding affinity and is predicted to increase the structural order of this region. Furthermore, MYCi selectively modulated the expression of the same genes regulated by lysine-acetylated MYC in cancer cells. These studies provide a rationale for selective targeting of acetylated, oncogenic MYC with small molecules.

cancer biology↗

bioRxiv2026

Mapping the Ligand-dependent Remodeling of the Conformational Entropy Landscape in Neurotensin Receptor 1 by NMR-guided Molecular Simulations

This study presents a comprehensive analysis of the dynamic properties and allosteric regulation mechanisms of Class A G protein-coupled receptors (GPCRs) by integrating molecular dynamics (MD) simulations with nuclear magnetic resonance (NMR) relaxation measurements. Utilizing generalized order parameters derived from NMR data and MD trajectories, we quantitatively assess conformational entropy changes that occur during receptor activation and ligand binding events. This approach enables a detailed characterization of protein flexibility at multiple timescales, revealing how dynamic fluctuations contribute to allosteric signal transmission within the receptor. Our results demonstrate that conformational entropy plays a pivotal role in modulating the functional states of Class A GPCRs, influencing the equilibrium between inactive and active conformations. By elucidating the interplay between structural dynamics and allostery, this work advances the molecular-level understanding of GPCR function and highlights the importance of entropy-driven effects in receptor signaling. The integrative methodology and findings provide a valuable framework for future investigations aimed at targeting receptor dynamics in drug discovery and rational design of allosteric modulators.

molecular biology↗

bioRxiv2026

The isolated Stachel peptide of the adhesion G protein-coupled receptor GPR126 is intrinsically disordered

Several members of the adhesion subfamily of G protein-coupled receptors (aGPCRs) are capable of self-activation by an internal agonist sequence (aka the Stachel) thats exposed upon removal or conformational changes of the N-terminal fragment of the receptor. Synthetic peptides derived from the Stachel sequence can be used as exogenous agonists. In the inactive form, the Stachel is sequestered as the {beta}13-strand within the GPCR Autoproteolysis-INducing (GAIN) domain, but it engages the seven transmembrane region as a helix when it is either an intramolecular sequence or a synthetic peptide. Little is known about the molecular details underlying this transition, but we hypothesize that a disordered conformation is central to this intermediate state in receptor activation. Despite the primarily helical Stachel AlphaFold3 and Pepfold4 models predicted with high confidence for the entire aGPCR subfamily, disorder predictions and biophysical experiments reveal a predominantly disordered conformation in solution. Investigating the ADGRG6/GPR126 Stachel peptide, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments reveal a predominantly random coil conformation in aqueous buffer, polar detergent micelles, and zwitterionic lipids. Titration of trifluoroethanol uncovered a two-state equilibrium between an unfolded and helix-containing conformation with NMR localizing a single-turn helix to residues L846-L849. Taken together, these data indicate the ADGRG6/GPR126 Stachel peptide is primarily disordered, but small populations may adopt a helix-containing conformer that seems to support a conformational-selection activation mechanism.

biophysics↗

bioRxiv2026

Chemical control of CSA geometry enables relaxation-optimized 19F-13C NMR probes

Fluorine NMR is a powerful tool for probing biomolecular structure and dynamics, yet the performance of 19F probes is fundamentally constrained by rapid transverse relaxation driven by chemical shift anisotropy (CSA). Despite its central role, CSA has largely been treated as an immutable nuclear property rather than a chemically addressable design parameter. Here we demonstrate that the geometry of the CSA tensor - specifically its magnitude, symmetry, and orientation relative to the internuclear dipolar interaction - constitutes a decisive and engineerable determinant of relaxation behavior in coupled 19F-13C spin systems. Guided by electronic-structure calculations and Bloch-Redfield-Wangsness relaxation theory, we establish quantitative design rules that predict when CSA-dipolar interference can be exploited to suppress transverse relaxation. Implementation of these principles in a cysteine-reactive fluoropyrimidine scaffold yields a reporter that supports simultaneous 19F and 13C TROSY optimization, validated by solid-state MAS NMR and protein-based experiments. When incorporated into the 42 kDa maltose binding protein, the probe exhibits exceptionally slow 13C transverse relaxation (R2 {approx} 2-3 s-1) corresponding to linewidths of [~]2 Hz that persist even at apparent molecular weights exceeding 200 kDa. These results recast relaxation optimization as a chemically programmable problem and provide a general framework for the rational design of next-generation NMR probes tailored to large, dynamic, and heterogeneous biomolecular systems.

biophysics↗

bioRxiv2025

Intrinsic conformational equilibria position arrestin-2 for activation

Arrestins regulate G protein-coupled receptor (GPCR) signaling by undergoing large-scale conformational rearrangements, yet the solution-state equilibria that underlie arrestin pre-activation remain poorly defined. Here, we use methyl-specific nuclear magnetic resonance spectroscopy, temperature-dependent chemical shift analysis, and relaxation measurements to characterize the intrinsic conformational landscape of full-length human arrestin-2 in solution. We identify two distinct equilibria with separable thermodynamic and kinetic signatures. A slow, enthalpically-favored process sensed by interdomain isoleucines I241 and I317 populates an active-like, interdomain-twisted conformation at physiological temperatures. In parallel, a faster, globally distributed equilibrium consistent with C-terminal tail release exhibits opposing thermodynamic behavior. Dynamic analyses reveal localized rigidification in the active-like minor states despite arrestins overall flexibility, while backbone relaxation data indicate widespread s-ms conformational exchange. Together, these results demonstrate that arrestin-2 intrinsically samples activation-relevant conformations in the absence of binding partners, providing a solution-state framework for arrestin pre-activation and signaling competence.

biophysics↗

bioRxiv2025

Stabilization versus flexibility: detergent-dependent trade-offs in neurotensin receptor 1 GPCR ensembles

Detergents provide essential membrane-mimetic environments for studying G protein-coupled receptors (GPCRs), but their molecular impact on receptor energetics remains incompletely understood. We combined ligand binding, thermostability measurements and atomistic molecular dynamics to dissect detergent- versus ligand-driven stabilization in a thermostabilized neurotensin receptor 1 (enNTS1). Circular dichroism and ligand binding assays revealed that apo enNTS1 becomes progressively more stable in decyl maltoside (DM), dodecyl maltoside (DDM), and lauryl maltose neopentyl glycol (LMNG). Yet this gain in baseline stability was accompanied by a paradox: LMNG, the most stabilizing detergent, supported the weakest neurotensin agonist binding affinity. Thermodynamic analysis resolved this contradiction by partitioning stability into detergent-driven conformational rigidity ({Delta}Gconf) and ligand-induced stabilization ({Delta}Gligand). In DM, {Delta}Gligand contributions were large, consistent with the receptors engineered background. In contrast, LMNG maximized {Delta}Gconf, constraining conformational flexibility and reducing {Delta}Gligand. Molecular dynamics simulations corroborated these results, showing that LMNG formed denser, less mobile detergent shells around the receptor, enhancing protein-detergent interaction energies while limiting conformational flexibility. Redistribution of ligand contacts, particularly at neurotensin residue Y11, further underscored detergent-dependent modulation of the binding pocket. These results highlight a fundamental trade-off: LMNG provides exceptional receptor stabilization, supporting structural studies, but may mask conformational states relevant to signaling. In contrast, less rigid detergents preserve ligand-induced transitions at the expense of stability. These findings emphasize that detergent choice should be guided by whether the goal is structural resolution or dynamic characterization.

biophysics↗

bioRxiv2025

Role of Uba7-UFD in E1-E2 ISGylation: NMR-Based Insights into E2 Binding Dynamics

The ISGylation pathway, a key post-translational modification, plays a pivotal role in the innate immune response by covalently attaching ISG15 to target proteins. This cascade involves a series of enzymatic steps, including activation by the E1 enzyme Uba7, conjugation by the E2 enzyme UbcH8, and ligation by an E3 ligase. Central to this process is the ubiquitin-fold domain (UFD) of Uba7, which facilitates the transfer of ISG15 to UbcH8. However, the structural and mechanistic details of this interaction remain poorly understood. In this work, we present the solution NMR structure and functional analysis of the ubiquitin-fold domain of human Uba7, the E1 enzyme in the ISGylation cascade. Through detailed NMR titration experiments and mutational studies, we mapped the interaction surface of Uba7-UFD with UbcH8, identifying key residues and their contributions to the E1-E2 interaction. We found that Uba7-UFD is flexible in its free form; this flexibility is conserved across ubiquitin-like systems but shows a regional shift that may play an important role in correct E2 and Ubl selection. Chemical shift perturbation and mutational analysis further demonstrate the importance of specific residues, particularly Cys996, in maintaining UFDs structural integrity and binding capacity. Additionally, mutations designed to alter the flexibility and length of the loop region between UFD and UbcH8 show significant effects on binding, indicating that these regions are crucial for efficient E2 recruitment. These findings provide new insights into the mechanistic basis of E2 enzyme selection in ISGylation and underscore the functional relevance of dynamic structural transitions in E1-E2 complex formation. Statement of SignificanceISGylation is a key ubiquitin-like modification pathway essential for antiviral defense, immune regulation, and protein quality control. However, the molecular principles that govern communication between the E1 and E2 enzymes in this pathway remain poorly defined. Here, we present the first NMR structure of the human Uba7 ubiquitin-fold domain (UFD) and its interaction with UbcH8. Our findings reveal how structural flexibility within the UFD is crucial for specific and efficient E2 recognition, providing fundamental insight into the dynamic mechanism of ISGylation and advancing our understanding of ubiquitin-like enzyme cascades relevant to cellular regulation and disease.

biochemistry↗

bioRxiv2024

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminus. Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the C-tail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and C-terminus (H8-Ctail) to investigate its structural stability, conformational dynamics and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement (PRE) NMR. Taken together, our studies reveal the H8C-tail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

biophysics↗

bioRxiv2023

Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease I (APE1) acts as both an endonuclease and a redox factor to ensure cell survival. The two activities require different conformations of APE1. As an endonuclease, APE1 is fully folded. As a redox factor, APE1 must be partially unfolded to expose the buried residue Cys65, which reduces transcription factors including AP-1, NF-{kappa}B, and HIF-1 and thereby enables them to bind DNA. To determine a molecular basis for partial unfolding associated with APE1s redox activity, we characterized specific interactions of a known redox inhibitor APX3330 with APE1 through waterLOGSY and 1H-15N HSQC NMR approaches using ethanol and acetonitrile as co-solvents. We find that APX3330 binds to the endonuclease active site in both co-solvents and to a distant small pocket in acetonitrile. Prolonged exposure of APE1 with APX3330 in acetonitrile resulted in a time-dependent loss of 1H-15N HSQC chemical shifts ([~]35%), consistent with partial unfolding. Regions that are partially unfolded include adjacent N- and C-terminal beta strands within one of the two sheets comprising the core, which converge within the small binding pocket defined by the CSPs. Removal of APX3330 via dialysis resulted in a slow reappearance of the 1H-15N HSQC chemical shifts suggesting that the effect of APX3330 is reversible. APX3330 significantly decreases the melting temperature of APE1 but has no effect on endonuclease activity using a standard assay in either co-solvent. Our results provide insights on reversible partial unfolding of APE1 relevant for its redox function as well as the mechanism of redox inhibition by APX3330. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/547112v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13f9fe2org.highwire.dtl.DTLVardef@19cbcborg.highwire.dtl.DTLVardef@1927c7aorg.highwire.dtl.DTLVardef@2a3cc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at E1 and E3 interfaces - providing hypotheses for the proteins functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.

biochemistry↗

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗

bioRxiv2022

Ligands tune the local and global motions of neurotensin receptor 1 (NTS1): a DFT-guided solution NMR analysis

Unlike many signaling proteins that function as binary switches between on and off states, G protein-coupled receptors (GPCRs) exhibit basal activity that can be increased or decreased by numerous ligands. A given receptor can recognize multiple ligands, allosteric modulators, and transducers to create a complex free energy landscape. Many of the lowest energy states have been captured by static structural techniques while detailing the wells widths, metastable states, and the transition between them, is still in its infancy. Nuclear magnetic resonance (NMR) spectroscopy can monitor the structure and dynamics of GPCR ensembles across fifteen orders-of-magnitude, but technical challenges have limited its application to super-microsecond timescales. Focusing on a prototypical peptide-binding GPCR, the neurotensin receptor 1 (NTS1), we employed NMR and density functional theory (DFT) to probe global sub-nanosecond motions. The near random coil chemical shifts of the apo receptor produced a poor correlation with theoretical predictions that may indicate a high degree of conformational averaging in solution, a crystallization artifact, or both. Whereas orthosteric agonists and antagonists both rigidified the receptor, but to varying degrees, which suggests conformational entropy differentially contributes to their respective pharmacology. The strong correlations of observed and theoretical chemical shifts lend confidence to interpreting spectra in terms of local structure, methyl dihedral angle geometry, and pico-second timescale transitions. Together, our results suggest a role for sub-nanosecond dynamics and conformational entropy in GPCR ligand discrimination.

biophysics↗

bioRxiv2022

A Method for Selective 19F-Labeling Absent of Probe Sequestration (SLAPS)

Fluorine (19F) offers several distinct advantages for biomolecular nuclear magnetic resonance (NMR) spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine-reactive 19F-probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein-coupled receptors (GPCRs). As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks - especially when analyzing systems with intermediate to slow timescale conformational exchange. Here, we uncovered a second source of offsite labeling: non-covalent probe sequestration by detergent micelles. We present a simple four-step protocol for Selective Labeling Absent of Probe Sequestration (SLAPS): physically-disrupt cell membranes in the absence of detergent, incubate membranes with cysteine-reactive 19F-probes, remove excess unreacted 19F-probe molecules via ultracentrifugation, and finally solubilize in the detergent of choice. SLAPS should be broadly applicable to other lipophilic cysteine-reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics.

biophysics↗

bioRxiv2021

The Effect of Ligands and Transducers on the Neurotensin Recep-tor 1 (NTS1) Conformational Ensemble

Using a discrete, intracellular 19F-NMR probe on transmembrane helix 6 (TM6) of the Neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptors structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the ms-s timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other GPCRs, the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to {beta}-arrestin-1 or the C-terminal helix of Gq, which comprises 60% of the GPCR/G protein interface surface area. Whereas {beta}-arrestin-1 exclusively selects for pre-existing active-like substates, the Gq peptide induces a new substate. Both transducer molecules promote substantial line-broadening of active-like states suggesting contributions from additional s-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and ii) the available static structures do not represent the entire conformational ensemble observed in solution.

biophysics↗

bioRxiv2021

An algebraic solution for determining overall rotational correlation times from cross-correlated relaxation rates

Accurate rotational correlation times ({tau}c) are critical for quantitative analysis of fast timescale NMR dynamics. As molecular weights increase, the classic derivation of{tau} c using transverse and longitudinal relaxation rates becomes increasingly unsuitable due to the non-trivial contribution of remote dipole-dipole interactions to longitudinal relaxation. Derivations using cross-correlated relaxation experiments, such as TRACT, overcome these limitations but are erroneously calculated in 65% of the citing literature. Herein, we developed an algebraic solutions to the Goldman relationship that facilitate rapid, point-by-point calculations for straightforward identification of appropriate spectral regions where global tumbling is likely to be dominant. The rigid-body approximation of the Goldman relationship has been previously shown to underestimate TRACT-based rotational correlation time estimates. This motivated us to develop a second algebraic solution that employs a simplified model-free spectral density function including an order parameter term that could, in principle, be set to an average backbone S2 {approx} 0.9 to further improve the accuracy of{tau} c estimation. These solutions enabled us to explore the boundaries of the Goldman relationship as a function of the H-N internuclear distance (r), difference of the two principal components of the axially-symmetric 15N CSA tensor ({Delta}{delta}N), and angle of the CSA tensor relative to the N-H bond vector ({theta}). We hope our algebraic solutions and analytical strategies will increase the accuracy and application of the TRACT experiment.

biophysics↗

bioRxiv2019

Molecular Basis for the Evolved Instability of a Human G-Protein Coupled Receptor

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Though they function at the plasma membrane, mammalian GnRHRs tend to accumulate within the secretory pathway. Their apparent instability is believed to have evolved in response to selection for attenuated GnRHR activity. Nevertheless, the structural basis of this adaptation remains unclear. We find that this adaptation coincides with a C-terminal truncation and an increase in the polarity of its transmembrane (TM) domains. This enhanced polarity compromises the translocon-mediated cotranslational folding of two TM domains. Moreover, replacing a conserved polar residue in TM6 with an ancestral hydrophobic residue partially restores GnRHR expression with minimal impact on function. An evolutionary analysis suggests variations in the polarity of this residue are associated with reproductive differences. Our findings suggest the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

biochemistry↗