John M Louis

The National Institute of Diabetes and Digestive and Kidney Diseases, 베서스다, Maryland, United States

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Publications (181)873.7 Total impact

  • Hoi Sung Chung · John M. Louis · Irina V. Gopich
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    ABSTRACT: 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 submicrosecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. 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.
    No preview · Article · Jan 2016 · The Journal of Physical Chemistry B
  • John M Louis · James L Baber · G Marius Clore
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    ABSTRACT: The conformational transition of the core domain of HIV-1 gp41 from a pre-hairpin intermediate to a six-helix bundle is responsible for virus-cell fusion. Several inhibitors have been designed that target the N-heptad repeat helical coiled-coil trimer that is fully accessible in the pre-hairpin intermediate. One such inhibitor is the peptide C34 derived from the C-heptad repeat of gp41 which forms the exterior of the six-helix bundle. Here, using a variety of biophysical techniques including dye tagging, size exclusion chromatography combined with multi-angle light scattering, double electron-electron resonance EPR spectroscopy and circular dichroism, we investigate the binding of C34 to two six-helix bundle mimetics comprising N- and C-heptad repeats either without (coreSP) or with (coreS) a short spacer connecting the two. In the case of coreSP, C34 directly exchanges with the C-HR. For coreS, up to two molecules of C34 bind the six-helix bundle via displacement of the C-heptad repeat. These results suggest that fusion inhibitors such as C34 can target a continuum of conformational states from the pre-hairpin intermediate to the six-helix bundle prior to the occurrence of irreversible fusion of viral and target cell membranes.
    No preview · Article · Oct 2015 · Biochemistry
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    ABSTRACT: Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the Translocase of the Outer Membrane (TOM) complex. Tom40 is the central subunit of the TOM complex and forms a pore in the mitochondrial outer membrane. To date the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N- and C-termini. To investigate Tom40 function, including the role of the N-terminal and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, including truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels, and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore neither the first 51 amino acids of the N-terminus nor the last 13 amino acids of the C-terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and, therefore, in transport function. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Sep 2015 · Journal of Biological Chemistry
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    Julien Roche · John M. Louis · Ad Bax · Robert B. Best
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    ABSTRACT: We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer (HIV-1 PR), 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 to 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. This article is protected by copyright. All rights reserved.
    Full-text · Article · Sep 2015 · Proteins Structure Function and Bioinformatics
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    ABSTRACT: N-terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multi-drug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67 and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the above properties and observe no strict correlation. A 10 to 35-fold decrease in cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by only ~4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 in TFR-PR20 affects the rate of autoprocessing more drastically (> 300-fold) compared to TFR-PR(PR20-3) because of the cumulative effect of eight non-cluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the above properties governing PR regulation and function.
    No preview · Article · Aug 2015 · Biochemistry
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    ABSTRACT: Although Kramers' theory for diffusive barrier crossing on a 1D free energy profile plays a central role in landscape theory for complex biomolecular processes, it has not yet been rigorously tested by experiment. Here we test this 1D diffusion scenario with single molecule fluorescence measurements of DNA hairpin folding. We find an upper bound of 2.5 μs for the average transition path time, consistent with the predictions by theory with parameters determined from optical tweezer measurements.
    No preview · Article · Jul 2015 · Physical Review Letters
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    ABSTRACT: 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.
    No preview · Article · May 2015 · Journal of Medicinal Chemistry
  • James L Baber · John M Louis · G Marius Clore
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    ABSTRACT: 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 the 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 found 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 was accompanied by a significant increase in Tm and effectively abolished the P(r) dependence on T. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Mar 2015 · Angewandte Chemie International Edition in English
  • James L. Baber · John M. Louis · G. Marius Clore
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    ABSTRACT: 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 the 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 found 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 was accompanied by a significant increase in Tm and effectively abolished the P(r) dependence on T.
    No preview · Article · Mar 2015 · Angewandte Chemie
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    ABSTRACT: 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 ecto-domain 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 (6HB) conforma-tion then involves competition between intermolecular interactions needed for formation of the symmetric 6HB 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 inter-molecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of tri-merization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.
    Full-text · Article · Jan 2015 · Journal of Biomolecular NMR
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    ABSTRACT: 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 (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB 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.
    Full-text · Article · Jan 2015 · Journal of Biomolecular NMR
  • Julien Roche · John M. Louis · Ad Bax

    No preview · Article · Jan 2015 · Biophysical Journal
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    Julien Roche · John M. Louis · Ad Bax
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    ABSTRACT: 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.
    Full-text · Article · Dec 2014 · ChemBioChem
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    ABSTRACT: 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.
    Full-text · Article · Aug 2014 · PLoS ONE
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    ABSTRACT: The molecular basis for high resistance to clinical inhibitors of HIV-1 protease (PR) was examined for the variant designated PRP51 that was selected for resistance to darunavir (DRV). High resolution crystal structures of PRP51 with the active site D25N mutation revealed a ligand free form and an inhibitor-bound form showing a unique binding site and orientation for DRV. This inactivating mutation is known to increase the dimer dissociation constant and DRV affinity of PR. The PRP51-D25N dimers were in the open conformation with widely separated flaps, as reported for other highly resistant variants. PRP51-D25N dimer bound two DRV molecules and showed larger separation of 8.7 Å between the closest atoms of the two flaps compared with 4.4 Å for the ligand-free structure of this mutant. The ligand-free structure, however, lacked van der Waals contacts between Ile50 and Pro81' from the other subunit in the dimer, unlike the majority of PR structures. DRV is bound inside the active site cavity, however, the inhibitor is oriented almost perpendicular to its typical position and exhibits only 2 direct hydrogen bond and two water-mediated interactions with atoms of PRP51-D25N compared with 11 hydrogen bond interactions seen for DRV bound in the typical position in wild-type enzyme. The atypical location of DRV may provide opportunities for design of novel inhibitors targeting the open conformation of PR drug resistant mutants.
    Full-text · Article · Apr 2014 · ACS Chemical Biology
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    ABSTRACT: 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.
    Full-text · Article · Feb 2014 · Proceedings of the National Academy of Sciences
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    Julien Roche · John M. Louis · Ad Bax

    Full-text · Article · Jan 2014 · Biophysical Journal
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    Full-text · Article · Jan 2014 · Biophysical Journal
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    Preview · Article · Jan 2014 · Biophysical Journal
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    ABSTRACT: A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.
    Full-text · Article · Nov 2013 · PLoS ONE

Publication Stats

7k Citations
873.70 Total Impact Points

Institutions

  • 1996-2016
    • The National Institute of Diabetes and Digestive and Kidney Diseases
      베서스다, Maryland, United States
  • 1988-2015
    • National Institutes of Health
      • • National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
      • • Laboratory of Chemical Physics (LCP)
      • • Laboratory of Experimental Gerontology (LEG)
      • • Laboratory of Cell and Developmental Biology
      • • Laboratory of Bioorganic Chemistry (LBC)
      • • Laboratory of Cell Biology
      베서스다, Maryland, United States
  • 2008
    • Georgia State University
      • Department of Chemistry
      Atlanta, Georgia, United States
  • 2003
    • National Eye Institute
      Maryland, United States
  • 1992
    • NCI-Frederick
      Фредерик, Maryland, United States
  • 1989-1990
    • National Cancer Institute (USA)
      Maryland, United States