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Fibrils formed by the 42-residue amyloid-β peptide (Aβ42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aβ42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-β subunit pairs within a single filament. Despite these differences, Aβ42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aβ42 fibril structures. Here we report two cryo-EM-based structures of Aβ42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aβ42 fibril samples. These results demonstrate that Aβ42 fibrils can exhibit a greater range of structural variations than seen in previous studies.

Understanding the catalytic mechanism of SARS-CoV-2 main protease (Mpro) can help in guiding drug design of specific small-molecule antivirals. A 2.0 Å resolution room-temperature X-ray crystal structure of inactive C145A mutant Mpro in complex with the octapeptide Ac-SAVLQSGF-CONH2 that resembles a Michaelis complex is reported., SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145–His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-β peptides (D,L-Aβ40 and D,L-Aβ42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (L-Aβ40 and L-Aβ42), suggesting a “chiral inactivation” approach to abrogating the neurotoxicity of Aβ oligomers (Aβ-CI). Here we report a structural study of D,L-Aβ40 fibrils, using electron microscopy, solid state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid state NMR spectra indicate molecular conformations in D,L-Aβ40 fibrils that resemble those in known L-Aβ40 fibril structures. However, quantitative measurements of (13)C-(13)C and (15)N-(13)C distances in selectively labeled D,L-Aβ40 fibril samples indicate a qualitatively different supramolecular structure. While cross-β structures in mature L-Aβ40 fibrils are comprised of in-register, parallel β-sheets, our data indicate antiparallel β-sheets in D,L-Aβ40 fibrils, with alternation of D and L molecules along the fibril growth direction, i.e., antiparallel “rippled sheet” structures. The solid state NMR data suggest coexistence of D,L-Aβ40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the β-strand segments identified by solid state NMR. These results provide insight into the structural basis for Aβ-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-racglycerol) (POPG):1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q. Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. et al. J. Am. Chem. Soc. 2016, 138, 5789–5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (ΔR(2)) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates (R(2,B)) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (~3 μs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160(660–674)) exists in a monomer-trimer equilibrium with an association constant in the micro-molar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D(2)O than H(2)O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, (13)C(α) chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination., Three-colour FRET is a powerful tool to study macromolecular conformational dynamics, but is temporally limited due to the experimental complexity. Here the authors develop experimental and analytical methods for probing submillisecond-time scale dynamics using single continuous-wave excitation.

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA–SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA–SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15 N and 13 C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15 N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15 N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N–H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA–SP1 cleavage.

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38 °C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal mol

Description, Heptad repeat regions of SARS-CoV-2 spike protein transiently disrupt membrane integrity while driving cellular entry., Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the intervening structural transition, important for the design of fusion inhibitors, has remained elusive. Using nuclear magnetic resonance spectroscopy and other biophysical methods, we demonstrate the presence of α-helical, membrane-bound, intermediate states of spike’s heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface while in a slow dynamic equilibrium with the postfusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure-activity relationship (SAR) study of noncovalent compounds that bind in the enzyme's substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor's P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter case, time-ordering of the NUS data points by the normalized radial length yields a reduction of sampling artifacts, regardless of the spectral reconstruction algorithm. The disadvantage of time-ordered NUS sampling is that halting the experiment prior to its completion will result in lower spectral resolution, rather than a sparser data matrix. Alternatively, digitally correcting for sample decay prior to reconstruction of randomly ordered NUS data points can mitigate reconstruction artifacts, at the cost of somewhat lower sensitivity. Application of these sampling schemes to the Alzheimer's amyloid beta (Aβ1-42) peptide at an elevated concentration, low temperature, and 3 kbar of pressure, where approximately 75% of the peptide reverts to an NMR-invisible state during the collection of a 3D 15N-separated NOESY spectrum, highlights the improvement in artifact suppression and reveals weak medium-range NOE contacts in several regions, including the C-terminal region of the peptide.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag–Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag–Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions., NMR studies define how the P182L mutation alters intramolecular interactions to increase HSP27 accessibility for numerous binding proteins, compromising its chaperone function in patient cell‐derived motor neurons.

Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66', results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66') provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66' asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66' subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.

Significance The root cause of pathology in sickle cell disease is the polymerization of the mutant hemoglobin S upon deoxygenation in the tissues to form fibers. Both the amount of fiber at equilibrium and the kinetics of fiber formation depend on the partial pressure of oxygen. We show that control of polymerization by oxygen at equilibrium can be better explained by a recent extension of the famous two-state allosteric model of Monod, Wyman, and Changeux. Because of the unusual kinetics, polymerization is far out of equilibrium, which explains why patients with the disease manage to survive, while those with high levels of fetal hemoglobin and those with sickle trait (the heterozygous condition) have relatively benign conditions., The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D

We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α(3)D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid, and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three inter-dye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

Efficient and accurate models to predict the fitness of a sequence would be extremely valuable in protein design. We have explored the use of statistical potentials for the coevolutionary fitness landscape, extracted from known protein sequences, in conjuction with Monte Carlo simulations, as a tool for design. As proof of principle, we created a series of predicted high-fitness sequences for three different protein folds, representative of different structural classes: the GA (all-α) and GB (α/β) binding domains of streptococcal protein G, and an SH3 (all-β) domain. We found that most of the designed proteins can fold stably to the target structure, and a structure for a representative of each for GA, GB and SH3 was determined. Several of our designed proteins were also able to bind to native ligands, in some cases with higher affinity than wild-type. Thus, a search using a statistical fitness landscape is an remarkably effective tool for finding novel stable protein sequences.

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and post-fusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. 15N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Forster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.

Brain tissue of Alzheimer’s disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aβ(40) and Aβ(42). However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (< 1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Aβ(40) exhibit a high degree of order, reflected by slow longitudinal (15)N relaxation (T(1) >5 s) for residues 18–21 and 31–34, whereas the N-terminal 10 residues relax much faster (T(1) ≤1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca. 1000 s(−1) after the oligomerization is initiated.

Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter case, time-ordering of the NUS data points by the normalized radial length yields a reduction of sampling artifacts, regardless of the spectral reconstruction algorithm. The disadvantage of time-ordered NUS sampling is that halting the experiment prior to its completion will result in lower spectral resolution, rather than a sparser data matrix. Alternatively, digitally correcting for sample decay prior to reconstruction of randomly ordered NUS data points can mitigate reconstruction artifacts, at the cost of somewhat lower sensitivity. Application of these sampling schemes to the Alzheimer’s amyloid beta (Aβ(1–42)) peptide at an elevated concentration, low temperature, and 3 kbar of pressure, where approximately 75% of the peptide reverts to an NMR-invisible state during the collection of a 3D (15)N-separated NOESY spectrum, highlights the improvement in artifact suppression and reveals weak medium-range NOE contacts in several regions, including the C-terminal region of the peptide.

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer’s disease by mitigating against intracellular β-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-β(1–42) (Aβ42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aβ42 with GroEL using a variety of biophysical techniques. Aβ42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high–molecular-weight polydisperse aggregates present in fresh soluble Aβ42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aβ42 reveals the presence of three anchor-binding regions (residues 16–21, 31–34, and 40–41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aβ42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aβ42 construct, indicating that Aβ42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aβ42 species and the appearance of Aβ42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aβ42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer’s disease.

In single-molecule Förster resonance energy transfer (FRET) spectroscopy, the dynamics of molecular processes are usually determined by analyzing the fluorescence intensity of donor and acceptor dyes. Since FRET efficiency is related to fluorescence lifetimes, additional information can be extracted by analyzing fluorescence intensity and lifetime together. For fast processes where individual states are not well separated in a trajectory, it is not easy to obtain the lifetime information. Here, we present analysis methods to utilize fluorescence lifetime information from single-molecule FRET experiments, and apply these methods to three fast-folding, two-state proteins. By constructing 2D FRET efficiency-lifetime histograms, the correlation can be visualized between the FRET efficiency and fluorescence lifetimes in the presence of the sub-microsecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. In order to determine the kinetics and lifetime parameters accurately, we used a maximum likelihood method. We found that acceptor blinking can lead to inaccurate parameters in the donor delay time analysis. This problem can be solved by incorporating acceptor blinking into a model. While the analysis of acceptor delay times is not affected by acceptor blinking, it is more sensitive to the shape of the delay time distribution resulting from a broad conformational distribution in the unfolded state.

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.

An extremely drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with two potent antiviral investigational inhibitors. GRL-5010A and GRL-4410A were designed to introduce hydrogen bond interactions with the flexible flaps of the PR by incorporating gem-difluorines and alkoxy, respectively, at the C4 position of the bis-THF of darunavir. PR20 provides an excellent model for high level resistance, since clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme. GRL-5010A and GRL-4410A show inhibition constants of 4.3 ± 7.0 and 1.7 ± 1.8 nM, respectively, for PR20, compared to the binding affinity of 41 ± 1 nM measured for darunavir. Crystal structures of PR20 in complexes with the two inhibitors confirmed the new hydrogen bond interactions with Gly 48 in the flap of the enzyme. The two new compounds are more effective than darunavir in inhibiting mature PR20 and show promise for further development of antiviral agents targeting highly resistant PR mutants.

Pulsed double electron-electron resonance (DEER) provides pairwise P(r) distance distributions in doubly spin-labeled proteins. We report that in protonated proteins P(r) is dependent on the length of the second echo period T owing to local environmental effects on spin-label phase memory relaxation time Tm. For the protein ABD, this effect results in a 1.4 Å increase in the P(r) maximum from T = 6 to 20 μs. Protein A has a bimodal P(r) distribution and the relative height of the shorter distance peak at T = 10 μs, the shortest value required to obtain a reliable P(r), is reduced by 40% relative to that obtained by extrapolation to T = 0. Our results indicate that data at a series of T values are essential for quantitative interpretation of DEER to determine the extent of the T dependence and to extrapolate the results to T= 0. Complete deuteration (99%) of the protein is accompanied by a significant increase in Tm and effectively abolishes the P(r) dependence on T.

The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr−Purcell−Meiboom−Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease−Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

Folding and unfolding rates for the ultrafast folding villin subdomain were determined from a photon-by-photon analysis of fluorescence trajectories in single molecule FRET experiments. One of the obstacles to measuring fast kinetics in single molecule fluorescence experiments is blinking of the fluorophores on a timescale that is not well separated from the process of interest. By incorporating acceptor blinking into a two-state kinetics model, we show that it is possible to extract accurate rate coefficients on the microsecond time scale for folding and unfolding using the maximum likelihood method of I.V. Gopich and A. Szabo. This method yields the most likely parameters of a given model that can reproduce the observed photon trajectories. The extracted parameters agree with both the decay rate of the donor-acceptor cross correlation function and the results of ensemble equilibrium and kinetic experiments using nanosecond laser temperature jump.

We demonstrate that alignment of a structured peptide or small protein solubilized in mixed phospholipid:detergent micelles or bicelles, when embedded in a compressed gel or liquid crystalline medium, can be altered by either changing the phospholipid aggregate shape, charge, or both together. For the hemagglutinin fusion peptide solubilized in bicelles, we show that bicelle shape and charge do not change its helical hairpin structure but impact its alignment relative to the alignment medium, both in charged compressed acrylamide gel and in liquid crystalline d (GpG). The method can be used to generate sets of residual dipolar couplings (RDCs) that correspond to orthogonal alignment tensors, and holds promise for high-resolution structural refinement and dynamic mapping of membrane proteins.

Fusion of viral and cellular membranes is elicited by the HIV-1 envelope glycoprotein gp120/gp41. The precursor gp160, encoded by the Env gene, is cleaved post-translationally into two chains, gp120 and gp41, which remain non-covalently associated as a homotrimer of heterodimers and form a spike on the viral surface.[1] Upon CD4 recognition, the gp120 subunit dissociates from gp41, which remains anchored through its C-terminal transmembrane helix (TM) in the viral membrane. After gp120 dissociation, the N-terminal fusion peptide of gp41 is exposed and can insert into the host cell membrane.[2] Because of its high sequence conservation and its accessibility to the humoral immune system,[3] gp41 represents an attractive drug target for antiviral therapy as well as a key player in vaccine research.[4, 5]

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti-HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

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.

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.

We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PR(S17)), selected to represent high resistance by machine learning on genotype-phenotype data. Three of five mutations in PR(S17) correlating with major drug resistance, M46L, G48V, and V82S, and five of 11 natural variations differ from the mutations in two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PR(S17), which forms a stable dimer (10 nM), is ∼10- and 2-fold less efficient in processing the Gag polyprotein than the wild type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PR(S17) flanked by the 56-amino acid transframe region (TFP-p6pol) at its N-terminus, which is impossible upon expression of an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PR(S17) and mature PR(S17). Resistance of PR(S17) to eight protease inhibitors (PIs) relative to PR (Ki) increases by 1.5-5 orders of magnitude from 0.01 to 8.4 μM. Amprenavir, darunavir, atazanavir, and lopinavir, the most effective of the eight PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC50 values ranging from ∼7.5 to 60 μM. Thus, this process, crucial for stable dimer formation, shows inhibition ∼200-800-fold weaker than that of the mature PR(S17). TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC50 = 15 μM); amprenavir shows a 2-fold increase in IC50 (∼15 μM), and atazanavir and lopinavir show increased IC50 values of42 and70 μM, respectively.

DNA hairpins are the simplest structures for investigating fundamental aspects of nucleic acid folding mechanisms. For hairpins exhibiting two-state behavior, all of the mechanistic information on how the hairpin folds is contained in the transition path, the rare event in single molecule trajectories when the free energy barrier between folded and unfolded states is actually crossed. In the only experimental study of transition paths in nucleic acids to date, Woodside and coworkers measured an upper bound of 50 μs for the average transition path times (TPT) of several nucleic acids, limited by the time resolution of their optical tweezer experiments (Neupane et al. Phys. Rev. Lett. 2012). Neupane et al. also reconstructed the free energy surface for an indirect determination of average transition path times from Szabo's analytical theory for diffusive barrier crossing. We used confocal single molecule FRET to study a DNA hairpin with 4 base pairs in the stem and 21 T's in the loop and that has a folding time of 310 μs in 500mM NaCl at 22oC. Maximum likelihood analysis of 780 transitions in photon trajectories yielded an upper bound of 2.5 μs for the average transition path time (Truex et al., Phys. Rev. Lett. 2015), compared to the value of ∼4 μs predicted by Neupane et al. from hairpins with 9-30 base pair stems and 4 T's in the loop, providing an important test of energy landscape theory. Current experiments are aimed at reducing blinking, a major obstacle to measuring transition path times, and at eventually characterizing structural changes during transition paths, which will provide a very demanding test of mechanisms predicted by both theoretical models and simulations.

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analog p2-NC, DRV and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35–37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap, and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analog relative to wild type enzyme. PR20 complexes with PIs exhibit expanded S2/S2′ pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug resistant HIV.