Search

Triploidy is very useful in both aquaculture and some cultivated plants as the induced sterility helps to enhance growth and product quality, as well as acting as a barrier against the contamination of wild populations by escapees. To use genetic information from triploids for academic or breeding purposes, an efficient and robust method to genotype triploids is needed. We developed such a method for genotype calling from SNP arrays, and we implemented it in the R package named GenoTriplo. Our method requires no prior information on cluster positions and remains unaffected by shifted luminescence signals. The method relies on starting the clustering algorithm with an initial higher number of groups than expected from the ploidy level of the samples, followed by merging groups that are too close to each other to be considered as distinct genotypes. Accurate classification of SNPs is achieved through multiple thresholds of quality controls. We compared the performance of GenoTriplo with that of fitPoly, the only published method for triploid SNP genotyping with a free software access. This was assessed by comparing the genotypes generated by both methods for a dataset of 1232 triploid rainbow trout genotyped for 38,033 SNPs. The two methods were consistent for 89% of the genotypes, but for 26% of the SNPs, they exhibited a discrepancy in the number of different genotypes identified. For these SNPs, GenoTriplo had >95% concordance with fitPoly when fitPoly genotyped better. On the contrary, when GenoTriplo genotyped better, fitPoly had less than 50% concordance with GenoTriplo. GenoTriplo was more robust with less genotyping errors. It is also efficient at identifying low-frequency genotypes in the sample set. Finally, we assessed parentage assignment based on GenoTriplo genotyping and observed significant differences in mismatch rates between the best and second-best couples, indicating high confidence in the results. GenoTriplo could also be used to genotype diploids as well as individuals with higher ploidy level by adjusting a few input parameters., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Roche et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In aquaculture, sterile triploids are commonly used for production as sterility gives them potential gains in growth, yields, and quality. However, they cannot be reproduced, and DNA parentage assignment to their diploid or tetraploid parents is required to estimate breeding values for triploid phenotypes. No publicly available software has the ability to assign triploids to their parents. Here, we updated the R package APIS to support triploids induced from diploid parents. First, we created new exclusion and likelihood tables that account for the double allelic contribution of the dam and the recombination that can occur during female meiosis. As the effective recombination rate of each marker with the centromere is usually unknown, we set it at 0.5 and found that this value maximizes the assignment rate even for markers with high or low recombination rates. The number of markers needed for a high true assignment rate did not strongly depend on the proportion of missing parental genotypes. The assignment power was however affected by the quality of the markers (minor allele frequency, call rate). Altogether, 96-192 SNPs were required to have a high parentage assignment rate in a real rainbow trout dataset of 1,232 triploid progenies from 288 parents. The likelihood approach was more efficient than exclusion when the power of the marker set was limiting. When more markers were used, exclusion was more advantageous, with sensitivity reaching unity, very low false discovery rate (<0.01), and excellent specificity (0.96-0.99). Thus, APIS provides an efficient solution to assign triploids to their diploid parents., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1 H- 15 N TROSY and 1 H- 13 C methyl TROSY NMR spectra combined with 15 N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Disrupted-in-schizophrenia-1 (DISC1) is a scaffolding protein that plays a pivotal role in orchestrating signaling pathways involved in neurodevelopment, neural migration, and synaptogenesis. Among those, it has recently been reported that the role of DISC1 in the Akt/mTOR pathway can shift from a global translational repressor to a translational activator in response to oxidative stress induced by arsenic. In this study we provide evidence that DISC1 can directly bind arsenic via a C-terminal cysteine motif (C-X-C-X-C). A series of fluorescence-based binding assays were conducted with a truncated C-terminal domain construct of DISC1 and a series of single, double, and triple cysteine mutants. We found that arsenous acid, a trivalent arsenic derivative, specifically binds to the C-terminal cysteine motif of DISC1 with low micromolar affinity. All three cysteines of the motif are required for high-affinity binding. Electron microscopy experiments combined with in silico structural predictions reveal that the C-terminal of DISC1 forms an elongated tetrameric complex. The cysteine motif is consistently predicted to be located within a loop, fully exposed to solvent, providing a simple molecular framework to explain the high-affinity of DISC1 toward arsenous acid. This study sheds light on a novel functional facet of DISC1 as an arsenic binding protein and highlights its potential role as both a sensor and translational modulator within Akt/mTOR pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Watanabe, Khu, Warren, Shin, Stewart and Roche.)

Dendrite morphogenesis is essential for neural circuit formation, yet the molecular mechanisms underlying complex dendrite branching remain elusive. Previous studies on the highly branched Caenorhabditis elegans PVD sensory neuron identified a membrane co-receptor complex that links extracellular signals to intracellular actin remodeling machinery, promoting high-order dendrite branching. In this complex, the claudin-like transmembrane protein HPO-30 recruits the WAVE regulatory complex (WRC) to dendrite branching sites, stimulating the Arp2/3 complex to polymerize actin. We report here our biochemical and structural analysis of this interaction, revealing that the intracellular domain (ICD) of HPO-30 is intrinsically disordered and employs two distinct mechanisms to regulate the actin cytoskeleton. First, HPO-30 ICD binding to the WRC requires dimerization and involves the entire ICD sequence, rather than a short linear peptide motif. This interaction enhances WRC activation by the GTPase Rac1. Second, HPO-30 ICD directly binds to the sides and barbed end of actin filaments. Binding to the barbed end requires ICD dimerization and inhibits both actin polymerization and depolymerization, resembling the actin capping protein CapZ. These dual functions provide an intriguing model of how membrane proteins can integrate distinct mechanisms to fine-tune local actin dynamics., Competing Interests: DK, HN, UP, RS, BN, JR, BC No competing interests declared, KS Reviewing editor, eLife, (© 2023, Kramer et al.)

Most transcription factors possess at least one long intrinsically disordered transactivation domain that binds to a variety of coactivators and corepressors and plays a key role in modulating the transcriptional activity. Despite the crucial importance of these domains, the structural and functional basis of transactivation remains poorly understood. Here, we focused on activating transcription factor 4 (ATF4)/cAMP response element-binding protein-2, an essential transcription factor for cellular stress adaptation. Bioinformatic sequence analysis of the ATF4 transactivation domain sequence revealed that the first 125 amino acids have noticeably less propensity for structural disorder than the rest of the domain. Using solution nuclear magnetic resonance spectroscopy complemented by a range of biophysical methods, we found that the isolated transactivation domain is predominantly yet not fully disordered in solution. We also observed that a short motif at the N-terminus of the transactivation domain has a high helical propensity. Importantly, we found that the N-terminal region of the transactivation domain is involved in transient long-range interactions with the basic-leucine zipper domain involved in DNA binding. Finally, in vitro phosphorylation assays with the casein kinase 2 show that the presence of the basic-leucine zipper domain is required for phosphorylation of the transactivation domain. This study uncovers the intricate coupling existing between the transactivation and basic-leucine zipper domains of ATF4, highlighting its potential regulatory significance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA , which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC , is found in all MB operons and, in conjunction with mbnB , is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC , a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the Δ mbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the Δ mbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme., Competing Interests: The authors declare no competing interest.

Disrupted-in-schizophrenia-1 ( DISC1 ) is a scaffold protein of significant importance for neuro-development and a prominent candidate protein in the etiology of mental disorders. In this work, we investigate the role of conformational heterogeneity and local structural disorder in the oligomerization pathway of the full-length DISC1 and of two truncation variants. Through extensive coarse-grained molecular dynamics simulations with a predictive energy landscape-based model, we shed light on the interplay of local and global disorder which lead to different oligomerization pathways. We found that both global conformational heterogeneity and local structural disorder play an important role in shaping distinct oligomerization trends of DISC1 . This study also sheds light on the differences in oligomerization pathways of the full-length protein compared to the truncated variants produced by a chromosomal translocation associated with schizophrenia. We report that oligomerization of full-length DISC1 sequence works in a nonadditive manner with respect to truncated fragments that do not mirror the conformational landscape or binding affinities of the full-length unit.

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.

Protein conformational landscapes define their functional properties as well as their proteostasis. Hence, detailed mapping of these landscapes is necessary to understand and modulate protein conformation. The combination of high pressure and NMR provides a particularly powerful approach to characterizing protein conformational transitions. First, pressure, because its effects on protein structure arise from elimination of solvent excluded void volume, represents a more subtle perturbation than chemical denaturants, favoring the population of intermediates. Second, the residue-specific and multifaceted nature of NMR observables informs on many local structural properties of proteins, aiding in the characterization of intermediate and excited states., (© 2019 Elsevier Inc. All rights reserved.)

In addition to encoding RNA primary structures, genomes also encode RNA secondary and tertiary structures that play roles in gene regulation and, in the case of RNA viruses, genome replication. Methods for the identification of functional RNA structures in genomes typically rely on scanning analysis windows, where multiple partially-overlapping windows are used to predict RNA structures and folding metrics to deduce regions likely to form functional structure. Separate structural models are produced for each window, where the step size can greatly affect the returned model. This makes deducing unique local structures challenging, as the same nucleotides in each window can be alternatively base paired. We are presenting here a new approach where all base pairs from analysis windows are considered and weighted by favorable folding. This results in unique base pairing throughout the genome and the generation of local regions/structures that can be ranked by their propensity to form unusually thermodynamically stable folds. We applied this approach to the Zika virus (ZIKV) and HIV-1 genomes. ZIKV is linked to a variety of neurological ailments including microcephaly and Guillain-Barré syndrome and its (+)-sense RNA genome encodes two, previously described, functionally essential structured RNA regions. HIV, the cause of AIDS, contains multiple functional RNA motifs in its genome, which have been extensively studied. Our approach is able to successfully identify and model the structures of known functional motifs in both viruses, while also finding additional regions likely to form functional structures. All data have been archived at the RNAStructuromeDB (www.structurome.bb.iastate.edu), a repository of RNA folding data for humans and their pathogens., Competing Interests: The authors declare that they have no competing interests.

Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence modulates the configurational landscape of proteins remains to be defined. Protein configurational equilibria are generally characterized by using various chemical denaturants or by changing temperature or pH. Another thermodynamic parameter which is less often used in such studies is high hydrostatic pressure. This review discusses the basis for pressure effects on protein structure and stability, and describes how the unique mechanisms of pressure-induced unfolding can provide unique insights into protein conformational landscapes., (© 2018 The Author(s).)

The development of computational efficient models is essential to obtain a detailed characterization of the mechanisms underlying the folding of proteins and the formation of amyloid fibrils. Structure-based computational models (Go-model) with Cα or all-atom resolutions have been able to successfully delineate the mechanisms of folding of several globular proteins and offer an interesting alternative to computationally intensive simulations with explicit solvent description. Here, we explore the limits of Go-model predictions by analyzing the folding of the nonglobular repeat domain proteins Notch Ankyrin and p16 INK4 and the formation of human islet amyloid polypeptide (hIAPP) fibrils. Folding trajectories of the repeat domain proteins revealed that an all-atom resolution is required to capture the folding pathways and cooperativity reported in experimental studies. The all-atom Go-model was also successful in predicting the free-energy landscape of hIAPP fibrillation, suggesting a "dock and lock" mechanism of fibril elongation. We finally explored how mutations can affect the co-assembly of hIAPP fibrils by simulating a heterogeneous system composed of wild-type and mutated hIAPP peptides. Overall, this study shows that all-atom Go-model-based simulations have the potential of discerning the effects of mutations and post-translational modifications in protein folding and association and may help in resolving the dichotomy between experimental and theoretical studies on protein folding and amyloid fibrillation.

Using fine-tuned hydrogen bonding criteria, a library of coiled peptide fragments has been generated from a large set of high-resolution protein X-ray structures. This library is shown to be an improved representation of ϕ/ψ torsion angles seen in intrinsically disordered proteins (IDPs). The ϕ/ψ torsion angle distribution of the library, on average, provides good agreement with experimentally observed chemical shifts and 3 J HN-Hα coupling constants for a set of five disordered proteins. Inspection of the coil library confirms that nearest-neighbor effects significantly impact the ϕ/ψ distribution of residues in the coil state. Importantly, 3 J HN-Hα coupling constants derived from the nearest-neighbor modulated backbone ϕ distribution in the coil library show improved agreement to experimental values, thereby providing a better way to predict 3 J HN-Hα coupling constants for IDPs, and for identifying locations that deviate from fully random behavior., (© 2017 The Protein Society. This article is a U.S. Government work and is in the public domain in the USA.)

High-pressure is a well-known perturbation method used to destabilize globular proteins. It is perfectly reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. In contrast to other perturbation methods such as heat or chemical denaturant that destabilize protein structures uniformly, pressure exerts local effects on regions or domains of a protein containing internal cavities. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility to monitor at a residue level the structural transitions occurring upon unfolding and to determine the kinetic properties of the process. High-pressure NMR experiments can now be routinely performed, owing to the recent development of commercially available high-pressure sample cells. This review summarizes recent advances and some future directions of high-pressure NMR techniques for the characterization at atomic resolution of the energy landscape of protein folding., (Copyright © 2017 Elsevier B.V. All rights reserved.)

High-pressure is a well-known perturbation method used to destabilize globular proteins and dissociate protein complexes or aggregates. The heterogeneity of the response to pressure offers a unique opportunity to dissect the thermodynamic contributions to protein stability. In addition, pressure perturbation is generally reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility of monitoring at an atomic resolution the structural transitions occurring upon unfolding and determining the kinetic properties of the process. The recent development of commercially available high-pressure sample cells greatly increased the potential applications for high-pressure NMR experiments that can now be routinely performed. This review summarizes the recent applications and future directions of high-pressure NMR techniques for the characterization of protein conformational fluctuations, protein folding and the stability of protein complexes and aggregates., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir., Competing Interests: The authors have declared that no competing interests exist.

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17-172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17-172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675-683) in the 17-172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17-172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17-172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17-172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.

Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism., (Published by Elsevier Ltd.)

A new and convenient method, named ARTSY-J, is introduced that permits extraction of the (3)JHNHα couplings in proteins from the relative intensities in a pair of (15)N-(1)H TROSY-HSQC spectra. The pulse scheme includes (3)JHNHα dephasing of the narrower TROSY (1)H(N)-{(15)N} doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where (3)JHNHα dephasing is suppressed. The effect of passive (1)H(α) spin flips downscales the apparent (3)JHNHα coupling by a uniform factor that depends approximately linearly on both the duration of the (3)JHNHα dephasing delay and the (1)H-(1)H cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and (1)H(α) spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3Hz. Measurement for the HIV-1 protease homodimer (22kDa) yields (3)JHNHα values that agree to better than 0.7Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual (3)JHNHα couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable ϕ angle information., (Published by Elsevier Inc.)

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ(1-40) and Aβ(1-42) into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ(1-42) compared to that of Aβ(1-40) are poorly understood. To explore in detail the structural propensity of the monomeric Aβ(1-40) and Aβ(1-42) peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ(1-40) and Aβ(1-42) peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JNCα, and (1)JNCα) recorded for Aβ(1-40) were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ(1-42), suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process.

Provided that care is taken in adjusting the WATERGATE element of a (1)H-(15)N TROSY-HSQC experiment, such that neither the water magnetization nor the (1)H(α) protons are inverted by its final 180° pulse, (3)JHNHα couplings can be measured directly from splittings in the (1)H dimension of the spectrum. With band-selective (1)H decoupling, very high (15)N resolution can be achieved. A complete set of (3)JHNHα values, ranging from 3.4 to 10.1 Hz was measured for the 56-residue third domain of IgG-binding protein G (GB3). Using the H-N-C(α)-H(α) dihedral angles extracted from a RDC-refined structure of GB3, (3)JHNHα values predicted by a previously parameterized Karplus equation agree to within a root-mean-square deviation (rmsd) of 0.37 Hz with the experimental data. Values measured for the Alzheimer's implicated Aβ(1-40) peptide fit to within an rmsd of 0.45 Hz to random coil (3)JHNHα values.

We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

The energetic and volumetric properties of a three-state protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure-dependent (15) N-relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (ΔV) and isothermal compressibilities (ΔκT ) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are 100 mL mol(-1) and 40 μL mol(-1)  bar(-1) , respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on-pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein. The compressibility of the intermediate is larger than that of the folded state reflecting the less rigid nature of the former relative to the latter., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6 HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6 HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. Although hundreds of crystal structures of protease-inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These latter structures reveal a large diversity of flap conformations, ranging from closed to semi-open to wide open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open, and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Defining the physical-chemical determinants of protein folding and stability, under normal and pathological conditions has constituted a major subfield in biophysical chemistry for over 50 years. Although a great deal of progress has been made in recent years towards this goal, a number of important questions remain. These include characterizing the structural, thermodynamic and dynamic properties of the barriers between conformational states on the protein energy landscape, understanding the sequence dependence of folding cooperativity, defining more clearly the role of solvation in controlling protein stability and dynamics and probing the high energy thermodynamic states in the native state basin and their role in misfolding and aggregation. Fundamental to the elucidation of these questions is a complete thermodynamic parameterization of protein folding determinants. In this chapter, we describe the use of high-pressure coupled to Nuclear Magnetic Resonance (NMR) spectroscopy to reveal unprecedented details on the folding energy landscape of proteins.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Application of band-selective homonuclear (BASH) (1)H decoupling pulses during acquisition of the (1)H free induction decay is shown to be an efficient procedure for removal of scalar and residual dipolar couplings between amide and aliphatic protons. BASH decoupling can be applied in both dimensions of a homonuclear 2D NMR experiment and is particularly useful for enhancing spectral resolution in the H(N)-H(α) region of NOESY spectra of peptides and proteins, which contain important information on the backbone torsion angles. The method then also prevents generation of zero quantum and Hz(N)-Hz(α) terms, thereby facilitating analysis of intraresidue interactions. Application to the NOESY spectrum of a hexapeptide fragment of the intrinsically disordered protein α-synuclein highlights the considerable diffusion anisotropy present in linear peptides. Removal of residual dipolar couplings between H(N) and aliphatic protons in weakly aligned proteins increases resolution in the (1)H-(15)N HSQC region of the spectrum and allows measurement of RDCs in samples that are relatively strongly aligned. The approach is demonstrated for measurement of RDCs in protonated (15)N/(13)C-enriched ubiquitin, aligned in Pf1, yielding improved fitting to the ubiquitin structure., (Published by Elsevier Inc.)

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone (1)H-(15)N, (15)N-(13)C', (1)H(α)-(13)C(α), and (13)C'-(13)C(α) one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with (1)H-(15)N and (1)H(α)-(13)C(α) RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in (13)C' chemical shift, (3)JHNHα values, and (13)C(α)-(13)C(β) RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.

The impact of pressure on the backbone (15) N, (1) H and (13) C chemical shifts in N-terminally acetylated α-synuclein has been evaluated over a pressure range 1-2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the (1) H(N) chemical shifts, which commonly is associated with the presence of low-lying "excited states", is much larger in α-synuclein than in model peptides. The linear pressure response of (1) H(N) chemical shift, commonly linked to H-bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of (13) C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in (3) JHN-Hα by 0.22 ± 0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N-terminally acetylated synuclein show a transient of approximately 15% population of α-helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions., (Copyright © 2012 Wiley Periodicals, Inc.)

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chatelier's principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the protein's conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys-66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys-66 is located. Above 2 kbar pressure at these pH values the protein with Lys-66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys-66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys-66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure., (Copyright © 2010 Wiley-Liss, Inc.)