Publications by authors named "James Bardwell"

94 Publications

A Comparative Analysis of Mucormycosis in Immunosuppressed Hosts Including Patients with Uncontrolled Diabetes in the Southwest United States.

Am J Med 2021 May 8. Epub 2021 May 8.

Division of Infectious Diseases.

Background: Mucormycosis (zygomycosis) is an invasive fungal infection that carries a high risk of morbidity and mortality. Uncontrolled diabetes mellitus and other immunocompromising conditions are risk factors for mucormycosis development. We here describe the differences in characteristics and outcomes of mucormycosis among solid organ transplant, hematological malignancy, and diabetes mellitus groups at our institution.

Methods: We conducted a retrospective chart review over the period of 2009-2020, with identifying patients using the International Classification of Diseases, Ninth and Tenth Revisions. Clinical, laboratory, and outcome data were collected.

Results: There were 28 patients identified: 7 solid organ transplant, 3 hematological malignancy, and 18 diabetes mellitus patients were included in the study. Three solid organ transplant patients experienced an episode of rejection, and another 3 had cytomegalovirus infection prior to presenting with mucormycosis. Four of seven solid organ transplant patients had a history of diabetes mellitus, but the median hemoglobin A1C was lower than in the diabetes mellitus group (6.3 vs 11.5; P = .006). The mortality rate difference between solid organ transplant and diabetes mellitus was not statistically significant: 2/7 (28.57%) vs 5/18 (27.78%); P = .66. Patients with bilateral disease (pulmonary or sinus) had significantly higher mortality (80% vs 13%, P = .008). There was no difference in mortality outcomes among the different types of antifungal therapies administered.

Conclusion: A multispecialty approach is imperative in mucormycosis therapy. While the underlying risk factors were different, the outcomes were comparable for the solid organ transplant and diabetes mellitus groups. Future larger and longitudinal studies are recommended.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.amjmed.2021.04.008DOI Listing
May 2021

Mechanism of the small ATP-independent chaperone Spy is substrate specific.

Nat Commun 2021 02 8;12(1):851. Epub 2021 Feb 8.

Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21120-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870927PMC
February 2021

Author Correction: A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

Nat Chem Biol 2021 Mar;17(3):360

Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-021-00756-zDOI Listing
March 2021

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

Nat Chem Biol 2021 03 11;17(3):344-350. Epub 2021 Jan 11.

Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA.

Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O, severely limiting its nicotine-degrading activity. Instead of using O as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term 'oxidase' in referring to members of the flavin-containing amine 'oxidase' family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-00712-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904663PMC
March 2021

A metabolite binding protein moonlights as a bile-responsive chaperone.

EMBO J 2020 10 3;39(20):e104231. Epub 2020 Sep 3.

Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019104231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560199PMC
October 2020

Increased surface charge in the protein chaperone Spy enhances its anti-aggregation activity.

J Biol Chem 2020 10 17;295(42):14488-14500. Epub 2020 Aug 17.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai, China

Chaperones are essential components of the protein homeostasis network. There is a growing interest in optimizing chaperone function, but exactly how to achieve this aim is unclear. Here, using a model chaperone, the bacterial protein Spy, we demonstrate that substitutions that alter the electrostatic potential of Spy's concave, client-binding surface enhance Spy's anti-aggregation activity. We show that this strategy is more efficient than one that enhances the hydrophobicity of Spy's surface. Our findings thus challenge the traditional notion that hydrophobic interactions are the major driving forces that guide chaperone-substrate binding. Kinetic data revealed that both charge- and hydrophobicity-enhanced Spy variants release clients more slowly, resulting in a greater "holdase" activity. However, increasing short-range hydrophobic interactions deleteriously affected Spy's ability to capture substrates, thus reducing its chaperone activity toward fast-aggregating substrates. Our strategy in chaperone surface engineering therefore sought to fine-tune the different molecular forces involved in chaperone-substrate interactions rather than focusing on enhancing hydrophobic interactions. These results improve our understanding of the mechanistic basis of chaperone-client interactions and illustrate how protein surface-based mutational strategies can facilitate the rational improvement of molecular chaperones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.012300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573262PMC
October 2020

Pulmonary Mucormycosis in a Heart Transplant Patient.

Am J Med 2020 09 19;133(9):e524-e525. Epub 2020 Mar 19.

University of Arizona, College of Medicine, Division of Infectious Diseases, Tucson.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.amjmed.2020.02.020DOI Listing
September 2020

Yeast Tripartite Biosensors Sensitive to Protein Stability and Aggregation Propensity.

ACS Chem Biol 2020 04 10;15(4):1078-1088. Epub 2020 Mar 10.

Department of Molecular, Cellular, and Developmental Biology and Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109-1085, United States.

In contrast to the myriad approaches available to study protein misfolding and aggregation , relatively few tools are available for the study of these processes in the cellular context. This is in part due to the complexity of the cellular environment which, for instance, interferes with many spectroscopic approaches. Here, we describe a tripartite fusion approach that can be used to assess protein stability and solubility in the cytosol of . Our biosensors contain tripartite fusions in which a protein of interest is inserted into antibiotic resistance markers. These fusions act to directly link the aggregation susceptibility and stability of the inserted protein to antibiotic resistance. We demonstrate a linear relationship between the thermodynamic stabilities of variants of the model folding protein immunity protein 7 (Im7) fused into the resistance markers and their antibiotic resistance readouts. We also use this system to investigate the properties of the yeast prion proteins Sup35 and Rnq1 and proteins whose aggregation is associated with some of the most prevalent neurodegenerative misfolding disorders, including peptide amyloid beta 1-42 (Aβ42), which is involved in Alzheimer's disease, and protein α-synuclein, which is linked to Parkinson's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.0c00083DOI Listing
April 2020

Identifying dynamic, partially occupied residues using anomalous scattering.

Acta Crystallogr D Struct Biol 2019 Dec 19;75(Pt 12):1084-1095. Epub 2019 Nov 19.

Department of Chemistry and Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA.

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798319014475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889914PMC
December 2019

SERF engages in a fuzzy complex that accelerates primary nucleation of amyloid proteins.

Proc Natl Acad Sci U S A 2019 11 28;116(46):23040-23049. Epub 2019 Oct 28.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109;

The assembly of small disordered proteins into highly ordered amyloid fibrils in Alzheimer's and Parkinson's patients is closely associated with dementia and neurodegeneration. Understanding the process of amyloid formation is thus crucial in the development of effective treatments for these devastating neurodegenerative diseases. Recently, a tiny, highly conserved and disordered protein called SERF was discovered to modify amyloid formation in and humans. Here, we use kinetics measurements and native ion mobility-mass spectrometry to show that SERF mainly affects the rate of primary nucleation in amyloid formation for the disease-related proteins Aβ40 and α-synuclein. SERF's high degree of plasticity enables it to bind various conformations of monomeric Aβ40 and α-synuclein to form structurally diverse, fuzzy complexes. This structural diversity persists into early stages of amyloid formation. Our results suggest that amyloid nucleation is considerably more complex than age-related conversion of Aβ40 and α-synuclein into single amyloid-prone conformations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1913316116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859325PMC
November 2019

Protein folding while chaperone bound is dependent on weak interactions.

Nat Commun 2019 10 23;10(1):4833. Epub 2019 Oct 23.

Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, 48109-1085, USA.

It is generally assumed that protein clients fold following their release from chaperones instead of folding while remaining chaperone-bound, in part because binding is assumed to constrain the mobility of bound clients. Previously, we made the surprising observation that the ATP-independent chaperone Spy allows its client protein Im7 to fold into the native state while continuously bound to the chaperone. Spy apparently permits sufficient client mobility to allow folding to occur while chaperone bound. Here, we show that strengthening the interaction between Spy and a recently discovered client SH3 strongly inhibits the ability of the client to fold while chaperone bound. The more tightly Spy binds to its client, the more it slows the folding rate of the bound client. Efficient chaperone-mediated folding while bound appears to represent an evolutionary balance between interactions of sufficient strength to mediate folding and interactions that are too tight, which tend to inhibit folding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-12774-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811625PMC
October 2019

Chaperone OsmY facilitates the biogenesis of a major family of autotransporters.

Mol Microbiol 2019 11 9;112(5):1373-1387. Epub 2019 Oct 9.

Howard Hughes Medical Institute and Department of Molecular, Cellular & Development Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

OsmY is a widely conserved but poorly understood 20 kDa periplasmic protein. Using a folding biosensor, we previously obtained evidence that OsmY has molecular chaperone activity. To discover natural OsmY substrates, we screened for proteins that are destabilized and thus present at lower steady-state levels in an osmY-null strain. The abundance of an outer membrane protein called antigen 43 was substantially decreased and its β-barrel domain was undetectable in the outer membrane of an osmY-null strain. Antigen 43 is a member of the diffuse adherence family of autotransporters. Like strains that are defective in antigen 43 production, osmY-null mutants failed to undergo cellular autoaggregation. In vitro, OsmY assisted in the refolding of the antigen 43 β-barrel domain and protected it from added protease. Finally, an osmY-null strain that expressed two members of the diffuse adherence family of autotransporters that are distantly related to antigen 43, EhaA and TibA, contained reduced levels of the proteins and failed to undergo cellular autoaggregation. Taken together, our results indicate that OsmY is involved in the biogenesis of a major subset of autotransporters, a group of proteins that play key roles in bacterial pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842399PMC
November 2019

Infection of Aortic Endograft Caused by Coccidioidomycosis.

Am J Med 2020 01 25;133(1):e1-e2. Epub 2019 Jul 25.

College of Medicine, Division of Infectious Diseases, University of Arizona, Tucson; College of Medicine, Valley Fever Center for Excellence, University of Arizona, Tucson.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.amjmed.2019.07.013DOI Listing
January 2020

Chaperone activation and client binding of a 2-cysteine peroxiredoxin.

Nat Commun 2019 02 8;10(1):659. Epub 2019 Feb 8.

Department of Molecular, Cellular and Developmental, University of Michigan, Ann Arbor, 48109-1085, MI, USA.

Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-08565-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368585PMC
February 2019

In vivo chloride concentrations surge to proteotoxic levels during acid stress.

Nat Chem Biol 2018 11 15;14(11):1051-1058. Epub 2018 Oct 15.

Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA.

To successfully colonize the intestine, bacteria must survive passage through the stomach. The permeability of the outer membrane renders the periplasm of Gram-negative bacteria vulnerable to stomach acid, which inactivates proteins. Here we report that the semipermeable nature of the outer membrane allows the development of a strong Donnan equilibrium across this barrier at low pH. As a result, when bacteria are exposed to conditions that mimic gastric juice, periplasmic chloride concentrations rise to levels that exceed 0.6 M. At these chloride concentrations, proteins readily aggregate in vitro. The acid sensitivity of strains lacking acid-protective chaperones is enhanced by chloride, suggesting that these chaperones protect periplasmic proteins both from acidification and from the accompanying accumulation of chloride. These results illustrate how organisms have evolved chaperones to respond to the substantial chemical threat imposed by otherwise innocuous chloride concentrations that are amplified to proteotoxic levels by low-pH-induced Donnan equilibrium effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-018-0143-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193267PMC
November 2018

Reply to 'Misreading chaperone-substrate complexes from random noise'.

Nat Struct Mol Biol 2018 11;25(11):990-991

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-018-0145-2DOI Listing
November 2018

Elaborating a coiled-coil-assembled octahedral protein cage with additional protein domains.

Protein Sci 2018 11 3;27(11):1893-1900. Epub 2018 Oct 3.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109.

De novo design of protein nano-cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetry-based strategy for protein assembly in which short, coiled-coil sequences mediate the assembly of a protein building block into a cage. The geometry of the cage is specified by the combination of rotational symmetries associated with the coiled-coil and protein building block. We have used this approach to design well-defined octahedral and tetrahedral cages. Here, we show that the cages can be further elaborated and functionalized by the addition of another protein domain to the free end of the coiled-coil: in this case by fusing maltose-binding protein to an octahedral protein cage to produce a structure with a designed molecular weight of ~1.8 MDa. Importantly, the addition of the maltose binding protein domain dramatically improved the efficiency of assembly, resulting in ~ 60-fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiled-coil motifs as off-the-shelf components to design MDa-sized protein cages to which additional structural or functional elements can be added in a modular manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.3497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201728PMC
November 2018

Periplasmic Chaperones and Prolyl Isomerases.

EcoSal Plus 2018 07;8(1)

Dept of Molecular Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109.

The biogenesis of periplasmic and outer membrane proteins (OMPs) in is assisted by a variety of processes that help with their folding and transport to their final destination in the cellular envelope. Chaperones are macromolecules, usually proteins, that facilitate the folding of proteins or prevent their aggregation without becoming part of the protein's final structure. Because chaperones often bind to folding intermediates, they often (but not always) act to slow protein folding. Protein folding catalysts, on the other hand, act to accelerate specific steps in the protein folding pathway, including disulfide bond formation and peptidyl prolyl isomerization. This review is primarily concerned with and periplasmic and cellular envelope chaperones; it also discusses periplasmic proline isomerization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/ecosalplus.ESP-0005-2018DOI Listing
July 2018

Electrostatic interactions are important for chaperone-client interaction in vivo.

Microbiology (Reading) 2018 07 5;164(7):992-997. Epub 2018 Jun 5.

Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA.

It has long been thought that chaperones are primarily attracted to their clients through the hydrophobic effect. However, in in vitro studies on the interaction between the chaperone Spy and its substrate Im7, we recently showed that long-range electrostatic interactions also play a key role. Spy functions in the periplasm of Gram-negative bacteria, which is surrounded by a permeable outer membrane. The ionic conditions in the periplasm therefore closely mimic those in the media, which allowed us to vary the ionic strength of the in vivo folding environment. Using folding biosensors that link protein folding to antibiotic resistance, we were able to monitor Spy chaperone activity in Escherichia coli in vivo as a function of media salt concentration. The chaperone activity of Spy decreased when the ionic strength of the media was increased, strongly suggesting that electrostatic forces play a vital role in the action of Spy in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mic.0.000676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152375PMC
July 2018

Selecting Conformational Ensembles Using Residual Electron and Anomalous Density (READ).

Methods Mol Biol 2018 ;1764:491-504

Department of Chemistry and Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA.

Heterogeneous and dynamic biomolecular complexes play a central role in many cellular processes but are poorly understood due to experimental challenges in characterizing their structural ensembles. To address these difficulties, we developed a hybrid methodology that combines X-ray crystallography with ensemble selections typically used in NMR studies to determine structural ensembles of heterogeneous biomolecular complexes. The method, termed READ, for residual electron and anomalous density, enables the visualization of heterogeneous conformational ensembles of complexes within crystals. Here we present a detailed protocol for performing the ensemble selections to construct READ ensembles. From a diverse pool of binding poses, a selection scheme is used to determine a subset of conformations that maximizes agreement with the X-ray data. Overall, READ is a general approach for obtaining a high-resolution view of dynamic protein-protein complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7759-8_31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148353PMC
February 2019

Folding against the wind.

Nat Chem Biol 2018 04;14(4):329-330

Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-018-0016-5DOI Listing
April 2018

Directed evolution to improve protein folding in vivo.

Curr Opin Struct Biol 2018 02 23;48:117-123. Epub 2017 Dec 23.

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA; Howard Hughes Medical Institute, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA. Electronic address:

Recently, several innovative approaches have been developed that allow one to directly screen or select for improved protein folding in the cellular context. These methods have the potential of not just leading to a better understanding of the in vivo folding process, they may also allow for improved production of proteins of biotechnological interest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2017.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880552PMC
February 2018

The Mechanism of HdeA Unfolding and Chaperone Activation.

J Mol Biol 2018 01 11;430(1):33-40. Epub 2017 Nov 11.

Department of Chemistry & Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA. Electronic address:

HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2017.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738273PMC
January 2018

Symmetry-Directed Self-Assembly of a Tetrahedral Protein Cage Mediated by de Novo-Designed Coiled Coils.

Chembiochem 2017 10 29;18(19):1888-1892. Epub 2017 Aug 29.

Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of protein-protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled-coil domains to assemble proteins into higher-order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well-defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled-coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled-coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201700406DOI Listing
October 2017

Folding while bound to chaperones.

Curr Opin Struct Biol 2018 02 19;48:1-5. Epub 2017 Jul 19.

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA; Howard Hughes Medical Institute, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA. Electronic address:

Chaperones are important in preventing protein aggregation and aiding protein folding. How chaperones aid protein folding remains a key question in understanding their mechanism. The possibility of proteins folding while bound to chaperones was reintroduced recently with the chaperone Spy, many years after the phenomenon was first reported with the chaperones GroEL and SecB. In this review, we discuss the salient features of folding while bound in the cases for which it has been observed and speculate about its biological importance and possible occurrence in other chaperones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2017.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775065PMC
February 2018

Chaperone-client interactions: Non-specificity engenders multifunctionality.

J Biol Chem 2017 07 15;292(29):12010-12017. Epub 2017 Jun 15.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109. Electronic address:

Here, we provide an overview of the different mechanisms whereby three different chaperones, Spy, Hsp70, and Hsp60, interact with folding proteins, and we discuss how these chaperones may guide the folding process. Available evidence suggests that even a single chaperone can use many mechanisms to aid in protein folding, most likely due to the need for most chaperones to bind clients promiscuously. Chaperone mechanism may be better understood by always considering it in the context of the client's folding pathway and biological function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.R117.796862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519353PMC
July 2017

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo.

J Vis Exp 2016 10 23(116). Epub 2016 Oct 23.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results. This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB's capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB's chaperone function in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/54527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092227PMC
October 2016

RNAs as chaperones.

RNA Biol 2016 12 28;13(12):1228-1231. Epub 2016 Oct 28.

a University of Michigan, Department of Molecular, Cellular, and Developmental Biology , Ann Arbor , MI , USA.

Recently, we found that RNA is a remarkably powerful chaperone that can bind to unfolded proteins and transfer them to Hsp70 for refolding. Combined with past studies on RNA-chaperone interactions, we propose a model for how chaperone RNA activity may contribute to the cellular response to stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15476286.2016.1247147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207374PMC
December 2016

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template.

J Mol Biol 2016 10 20;428(21):4361-4377. Epub 2016 Sep 20.

Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen DK-2200, Denmark.

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2016.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242314PMC
October 2016