Papers by Walter Englander
Social Science Research Network, 2020
The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early... more The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson's disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KD app = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its colocalization. Ubiquitin-enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking and transport associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism. .
medRxiv (Cold Spring Harbor Laboratory), Feb 23, 2023
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
Journal of Biological Chemistry, Sep 1, 2021
The superfamily of massively large AAA+ protein molecular machines functions to convert the chemi... more The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input “open” state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured “closed” state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.
Biochemistry, Jan 10, 1989
Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive res... more Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.
Archives of Biochemistry and Biophysics, May 1, 1957
Elsevier eBooks, 1968
Publisher Summary This chapter discusses the measurement of hydrogen exchange (HX) kinetics of nu... more Publisher Summary This chapter discusses the measurement of hydrogen exchange (HX) kinetics of nucleic acids by using the Sephadex and dialysis methods. The movement of H between nucleic acid and H 2 O solvent is followed with tritium (T) tag. Nucleic acid is initially incubated in THO solvent so that its exchangeable hydrogens come to equilibrium with free, solvent T and H. Unbound T is then removed by Sephadex columns or rapid dialysis to initiate a unidirectional exchange out of bound T. To measure the amount of T remaining bound to the nucleic acid at any time, the T that has exchanged out during the test time interval is removed. In a series of samples taken as a function of time of exchanging out, the ratio of (bound) T level to nucleic acid content is measured, and a plot of these data trace the exchange out kinetics of the nucleic acid. The chapter discusses a rapid dialysis technique that can be used to remove THO from tritiated nucleic acid. The technique, then, is too slow for most experiments with DNA but can be useful for soluble RNA (sRNA) and ribosomes. HX experiments can give information on the amount of structure in nucleic acids in terms of the total number of slowly exchanging hydrogens present. Nucleic acid HX rates are determined by localized opening–closing reactions that, it now seems, are experienced by all macromolecules. These opening–closing reactions may be of basic importance for the functions that nucleic acid molecules perform and for the effect on these functions of various chemical and physical agents.
Biophysical Journal, Feb 1, 1991
The important role played by chemical exchange in solving the proton assignment problem for oxidi... more The important role played by chemical exchange in solving the proton assignment problem for oxidized and reduced horse cytochrome c is described. Some novel approaches for establishing oxidation-reduction exchange correlations in combinations of several two-dimensional spectra were used. Unambiguous chemical exchange correlations were established for 55 NH-C0H resonances and all the aromatic and side chain methyl resonances. Consistent although not fully unambiguous main chain proton correlations were observed for 47 of the remaining 49 residues. The many exchange correlations found serve to multiply cross-connect the two extensive, individually self-consistent networks of assignments found for the oxidized and reduced forms, and thus help to confirm both sets of assignments.
Journal of Biological Chemistry, Nov 1, 1980
ABSTRACT
Arteriosclerosis, Thrombosis, and Vascular Biology, Sep 1, 2021
Plasma triglycerides (TGs) are an independent predictor of the risk for CAD, the leading cause of... more Plasma triglycerides (TGs) are an independent predictor of the risk for CAD, the leading cause of death worldwide. TGs are also positively associated with risk and severity of hyperTG-induced acute pancreatitis (HTG-AP). Current therapies are often insufficient in reducing extremely elevated TGs. We believe that apolipoprotein A-V (apoA-V, encoded by APOA5) can fill this unmet medical need. ApoA-V is a potent modulator of TG metabolism; it enhances lipoprotein lipase TG hydrolysis. We hypothesize that naturally occurring human APOA5 variants can inform ApoA-V function and identify novel ApoA-V based therapeutic axes. We used the Penn Medicine Biobank (PMBB) to identify and measure plasma TGs of carriers of APOA5 variants predicted to change ApoA-V structure-function. Then, we used hydrogen-deuterium exchange mass spectroscopy to determine the secondary structure of ApoA-V, thereby identifying putative functional domains onto which we mapped our variants of interest. Finally, we characterized the plasma lipid effects of these mutants using adeno-associated viral (AAV) vectors in apoa5 knockout (KO) mice. We identified APOA5 variants associated with changes in plasma TGs. These variants primarily fall near the central heparin binding domain or C-terminal lipid binding domain. We selected APOA5 Q275X, which removes the entire lipid binding domain, for further interrogation. Apoa5 KO mice that received APOA5 Q275X AAV had higher plasma TGs than mice treated with WT APOA5 AAV. While WT ApoA-V protein associated with VLDL and HDL particles, Q275X ApoA-V protein appeared in lipoprotein free fractions. We have identified APOA5 variants associated with plasma TG phenotypes in humans, and mapped them to an experimentally determined ApoA-V secondary structure to identify the functional domains likely impacted. We have identified APOA5 Q275X as a loss of function variant that fails to bind lipoprotein particles and is associated with elevated plasma TGs. Continued study of this and other interesting naturally occurring variants will provide insight into the function of ApoA-V in TG metabolism. These insights can help us to therapeutically enhance ApoA-V to rapidly reduce TG levels during acute HTG-AP and to help prevent recurrent HTG-AP.
Science, Nov 5, 1993
Exploring the "Anfinsen trail," the pathway by which unstructured proteins spontaneously fold to ... more Exploring the "Anfinsen trail," the pathway by which unstructured proteins spontaneously fold to their native functional form, has become a central goal of the protein chemists. In order to understand a biochemical pathway, one wants to isolate and describe the intermediate structures that lead from precursor to product. Unfortunately the intermediate forms that define protein folding pathways cannot be isolated or studied by the usual methodologies, however powerful, because folding intermediates are by definition unstable.
Elsevier eBooks, 1972
ABSTRACT
Biochemistry, Sep 1, 1985
Two-dimensional nuclear magnetic resonance techniques were used to assign the N H , C,H, and C,H ... more Two-dimensional nuclear magnetic resonance techniques were used to assign the N H , C,H, and C,H protons of over 60 of the 104 amino acid residues in the 'H N M R spectrum of horse ferrocytochrome c. The majority of these amino acids were completely assigned. Assignments were based on the analysis of two-dimensional J-correlated (COSY), nuclear Overhauser effect (NOESY), and relayed COSY spectra and on comparisons of the J-correlated spectra of various cytochrome c species. Spin diffusion is not a problem with monomeric proteins the size of cytochrome c. Here these advances are illustrated with data that lead to the assignment of the heme-associated residues cysteine-14 and tryptophan-59, the axial ligands methionine-80 and histidine-18, the entire N-terminal helix, and several other amino acid spin systems. With these approaches, structure, structure change, the internal dynamics of cytochrome c, and the interaction of these with function are being studied, especially by observation of the hydrogen exchange behavior of 'This work was supported by NIH Grants AM 11295 and GM 31847. A.J.W. gratefully acknowledges a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship.
Protein Science, Jun 11, 2012
To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbo... more To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbone amide hydrogens of Staphylococcal nuclease were measured by NMR methods. A modified analysis was used to improve accuracy for the faster hydrogens. HX rates of both near surface and well buried hydrogens are spread over more than 7 orders of magnitude. These results were compared with previous hypotheses for HX rate determination. Contrary to a common assumption, proximity to the surface of the native protein does not usually produce fast exchange. The slow HX rates for unprotected surface hydrogens are not well explained by local electrostatic field. The ability of buried hydrogens to exchange is not explained by a solvent penetration mechanism. The exchange rates of structurally protected hydrogens are not well predicted by algorithms that depend only on local interactions or only on transient unfolding reactions. These observations identify some of the present difficulties of HX rate prediction and suggest the need for returning to a detailed hydrogen by hydrogen analysis to examine the bases of structure-rate relationships, as described in the companion paper (
Proteins, Apr 1, 1996
To determine when seconda r y structure forms as two chains coalesce to form an a-helical dimer, ... more To determine when seconda r y structure forms as two chains coalesce to form an a-helical dimer, the folding rates of variants of the coiled coil region of GCN4 were compared. Residues at non-perturbing positions along the exterior length of the helices were substituted one at a time with alanine and glycine to vary helix propensity and therefore h e r stability. For all variants, the bimolecular folding rate remains largely unchanged; the unfolding rate changes to largely account for the change in stability. Thus, contrary to most folding models, widespread helix is not yet formed at the rate-limiting step in the folding pathway. The high-energy transition state is a collapsed form that contains little if any secondary structure, as suggested for the globular protein cytochrome c (Sosnick et al., Proteins 24:413-426,1996).
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Papers by Walter Englander