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1. Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance.

Author

Solmaz Sobhanifar, Liam J Worrall, Dustin T King, Gregory A Wasney, Lars Baumann, Robert T Gale, Michael Nosella, Eric D Brown, Stephen G Withers, and Natalie C J Strynadka

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

Published
2016
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2. Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus

Author

J. Andrew N. Alexander, Liam J. Worrall, Jinhong Hu, Marija Vuckovic, Nidhi Satishkumar, Raymond Poon, Solmaz Sobhanifar, Federico I. Rosell, Joshua Jenkins, Daniel Chiang, Wesley A. Mosimann, Henry F. Chambers, Mark Paetzel, Som S. Chatterjee, and Natalie C. J. Strynadka

Subjects
Multidisciplinary
Abstract

Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses β-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (β-lactamase PC1) and mecA (β-lactam-resistant cell-wall transpeptidase PBP2a) expression3–7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1—the wild type and an autocleavage-deficient F284A mutant, with or without β-lactam—reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from β-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action—in this case, antibiotic resistance—through the direct cleavage of a repressor.

Published
2023

3. Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus

Author

J Andrew N, Alexander, Liam J, Worrall, Jinhong, Hu, Marija, Vuckovic, Nidhi, Satishkumar, Raymond, Poon, Solmaz, Sobhanifar, Federico I, Rosell, Joshua, Jenkins, Daniel, Chiang, Wesley A, Mosimann, Henry F, Chambers, Mark, Paetzel, Som S, Chatterjee, and Natalie C J, Strynadka

Abstract

Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden

Published
2022

5. The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates

Author

Krithika Muthuraman, Richard E. Higgs, Carl L. Hansen, Maren de Vries, Mark J. Mulligan, Kizzmekia S. Corbett, Ping Xiang, Nicole V. Johnson, Jory A. Goldsmith, Jason S. McLellan, John R. Mascola, Marissa H. Piper, Lucas Kraft, Jamie L. Blackbourne, Samuel J. Hinshaw, Davide Pellacani, Stefanie Žentelis, Christopher M. Wiethoff, Solmaz Sobhanifar, David R. Martinez, Franz J. Triana, Thomas P. Cujec, Barney S. Graham, Robert W. Cross, Ching-Lin Hsieh, Beverly A. Heinz, Denisa Foster, Ralph S. Baric, Sean A. Tycho, Jorg Hendle, David Collins, Eun Sung Yang, Ester Falconer, Daniel Wrapp, Andrew C. Adams, Roza Bidshahri, A. Pustilnik, Yuri Hwang, Adil Mohamed, Julian Davies, Yi Zhang, Patricia Brown-Augsburger, Bryan Edward Jones, Shawn J. Berens, Kevin R. Jepson, Bryan C. Barnhart, Kathryn Westendorf, Lingshu Wang, Viktoriya Borisevich, Meike Dittmann, Maia A. Smith, Deepa Balasubramaniam, Thomas W. Geisbert, Rodrigo Goya, Hayley M. Belli, Olubukola M. Abiona, and Marie I. Samanovic

Subjects
Antibodies, Viral, Article, 03 medical and health sciences, 0302 clinical medicine, Pharmacokinetics, medicine, Animals, Respiratory system, Neutralizing antibody, 030304 developmental biology, 0303 health sciences, biology, business.industry, SARS-CoV-2, COVID-19, General Medicine, biology.organism_classification, Virology, Antibodies, Neutralizing, Macaca mulatta, 3. Good health, Vaccination, Rhesus macaque, medicine.anatomical_structure, Viral replication, 030220 oncology & carcinogenesis, Spike Glycoprotein, Coronavirus, biology.protein, Antibody, business, Respiratory tract
Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Published
2020

6. LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection

Author

Mark J. Mulligan, Kizzmekia S. Corbett, Marissa H. Piper, Nicole V. Johnson, Jory A. Goldsmith, Roza Bidshahri, Carl L. Hansen, Robert W. Cross, Daniel Wrapp, A. Pustilnik, Yuri Hwang, Julian Davies, Adil Mohamed, Kathryn Westendorf, Beverly A. Heinz, Olubukola M. Abiona, Lucas Kraft, Jamie L. Blackbourne, Andrew C. Adams, Marie I. Samanovic, Samuel J. Hinshaw, Bryan Edward Jones, Sean A. Tycho, Christopher M. Wiethoff, Shawn J. Berens, Krithika Muthuraman, Stefanie Zentelis, Thomas W. Geisbert, Richard E. Higgs, Ralph S. Baric, Lingshu Wang, Jorg Hendle, Maia A. Smith, Hayley M. Belli, Meike Dittmann, Deepa Balasubramaniam, Solmaz Sobhanifar, Rodrigo Goya, Denisa Foster, John R. Mascola, Viktoriya Borisevich, Thomas P. Cujec, Barney S. Graham, Ping Xiang, Jason S. McLellan, Davide Pellacani, David R. Martinez, Maren de Vries, Patricia Brown-Augsburger, Franz J. Triana, David Collins, Ester Falconer, Kevin R. Jepson, Bryan C. Barnhart, and Ching-Lin Hsieh

Subjects
Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Medicine, skin and connective tissue diseases, Neutralizing antibody, Research Articles, biology, business.industry, fungi, Antiviral antibody, respiratory system, biology.organism_classification, Virology, respiratory tract diseases, STM r-articles, body regions, Coronavirus, Rhesus macaque, medicine.anatomical_structure, Viral replication, biology.protein, Antibody, business, Research Article, Respiratory tract
Abstract

LY-CoV555, a SARS-CoV-2 spike protein–specific antibody, neutralizes SARS-CoV-2 and protects the airways of nonhuman primates against infection., A monoclonal for SARS-CoV-2 Among the most promising therapeutic options for individuals with coronavirus disease 2019 (COVID-19) are monoclonal antibodies (mAbs). In this study, Jones et al. identified, characterized, and tested one such mAb, LY-CoV555, in vitro and in vivo. They found that LY-CoV555 bound to the severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) spike protein and prevented its interaction with angiotensin-converting enzyme 2. Prophylactic treatment with LY-CoV555 protected the upper and lower respiratory tracts of nonhuman primates from becoming infected with SARS-CoV-2. Together, these data support the clinical use of LY-CoV555 for treating patients with COVID-19., Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour−1 kg−1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Published
2020

7. One ring to rule them all: Current trends in combating bacterial resistance to the β-lactams

Author

Natalie C. J. Strynadka, Solmaz Sobhanifar, and Dustin T. King

Subjects
0301 basic medicine, medicine.drug_class, 030106 microbiology, Arms race, Antibiotics, Biology, Biochemistry, Microbiology, 03 medical and health sciences, 030104 developmental biology, Multidrug resistant bacteria, Antibiotic resistance, Political economy, β lactams, medicine, Molecular Biology, Beta-Lactamase Inhibitors
Abstract

From humble beginnings of a contaminated petri dish, β-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an "arms race" with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever-dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in β-lactam research. Here the current state of β-lactam antibiotics from a structural perspective was reviewed.

Published
2016

9. Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

Author

Stephen G. Withers, Solmaz Sobhanifar, Lars Baumann, Eric D. Brown, Robert T. Gale, Gregory A. Wasney, Natalie C. J. Strynadka, Michael Nosella, Liam J. Worrall, and Dustin T. King

Subjects
0301 basic medicine, Staphylococcus, Petroleum Products, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, Bacterial cell structure, Mass Spectrometry, chemistry.chemical_compound, Protein structure, Cell Wall, Biology (General), Pathology and laboratory medicine, Teichoic acid, Crystallography, biology, Protein Stability, Physics, Medical microbiology, Condensed Matter Physics, Enzyme structure, 3. Good health, Enzymes, Petroleum, Staphylococcus aureus, Physical Sciences, Crystal Structure, Organic Materials, Pathogens, Research Article, Methicillin-Resistant Staphylococcus aureus, QH301-705.5, 030106 microbiology, Immunology, Protein domain, Materials Science, Microbiology, Cell wall, 03 medical and health sciences, Protein Domains, Bacterial Proteins, Transferases, Virology, Microbial Control, Genetics, medicine, Solid State Physics, Protein Structure, Quaternary, Molecular Biology, Medicine and health sciences, Pharmacology, Biology and life sciences, Bacteria, Organisms, Active site, Glycosyltransferases, Proteins, RC581-607, Microbial pathogens, Teichoic Acids, 030104 developmental biology, chemistry, Antibiotic Resistance, Enzyme Structure, biology.protein, Enzymology, Tar, Parasitology, Bacterial pathogens, Methicillin Resistance, Antimicrobial Resistance, Immunologic diseases. Allergy, Chromatography, Liquid
Abstract

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase., Author Summary Historically, β-lactam class antibiotics such as methicillin have been very successful in the treatment of bacterial infections, effectively destroying bacteria by rupturing their cell walls while posing little harm to the human organism. In recent years, however, the alarming emergence of Methicillin Resistant S. aureus or MRSA has resulted in a world-wide health crisis, calling on new strategies to combat pathogenesis and antibiotic resistance. As such, understanding the pathways and players that orchestrate resistance is important for overcoming these mechanisms and restoring our powerful β-lactam antibiotic arsenal. In this article we describe the crystal structure of TarS, an enzyme responsible for the glycosylation of wall teichoic acid polymers of the S. aureus cell wall, a process that has been shown to be specifically responsible for methicillin resistance in MRSA. TarS is therefore a promising drug target whose inhibition in combinational therapies would result in MRSA re-sensitization to β-lactam antibiotics. Here we present the first structure of TarS together with several snap-shots of its substrate/product complexes, and elucidate important catalytic features that are valuable for rational drug design efforts to combat resistance in MRSA.

Published
2016

10. Fortifying the wall: synthesis, regulation and degradation of bacterial peptidoglycan

Author

Dustin T. King, Natalie C. J. Strynadka, and Solmaz Sobhanifar

Subjects
Glycosylation, Bacteria, Microbial metabolism, Morphogenesis, Peptidoglycan, Biology, Bacterial growth, Bacterial cell structure, Cell biology, Cell wall, chemistry.chemical_compound, Metabolic balance, chemistry, Biochemistry, Cell Wall, Structural Biology, Degradation (geology), Molecular Biology
Abstract

Growth and maintenance of the protective peptidoglycan cell wall are vital to bacterial growth and morphogenesis. Thus, the relative rate and spatiotemporal control of the synthesis and degradation of this net-like polymer defines bacterial cell shape. In recent years, our understanding of the processes that govern this delicate metabolic balance has improved and should lend insight into how to therapeutically target the system in the future.

Published
2013

11. Structural investigation of the C-terminal catalytic fragment of presenilin 1

Author

Krzysztof Mlynarczyk, Frank Löhr, Volker Dötsch, Frank Bernhard, Daniel Gottstein, Peter Güntert, Michal Kolinski, Birgit Schneider, Slawomir Filipek, Wojciech Pulawski, Solmaz Sobhanifar, Teppei Ikeya, and Umesh Ghoshdastider

Subjects
Proteases, Stereochemistry, Lipid Bilayers, Molecular Sequence Data, 010402 general chemistry, Cleavage (embryo), 01 natural sciences, Protein Structure, Secondary, Presenilin, 03 medical and health sciences, Presenilin-1, Humans, Amino Acid Sequence, Senile plaques, Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular, Peptide sequence, Micelles, 030304 developmental biology, 0303 health sciences, Multidisciplinary, Chemistry, Biological Sciences, Transmembrane protein, Protein Structure, Tertiary, 0104 chemical sciences, Transmembrane domain, Biocatalysis, Hydrophobic and Hydrophilic Interactions
Abstract

The γ-secretase complex has a decisive role in the development of Alzheimer’s disease, in that it cleaves a precursor to create the amyloid β peptide whose aggregates form the senile plaques encountered in the brains of patients. Γ-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer’s disease are linked to presenilin 1, the catalytic component of γ-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.

Published
2010

12. Reduced Expression of Hypoxia-Inducible Factor-1α in Perinecrotic Regions of Solid Tumors

Author

Eric J. Stanbridge, Peggy L. Olive, Solmaz Sobhanifar, and Christina Aquino-Parsons

Subjects
Cancer Research, Pathology, medicine.medical_specialty, Biopsy, Transplantation, Heterologous, Uterine Cervical Neoplasms, Mice, SCID, Astrocytoma, Biology, Mice, Necrosis, Antigens, Neoplasm, Cell Line, Tumor, Neoplasms, Gene expression, Biomarkers, Tumor, Image Processing, Computer-Assisted, medicine, Animals, Humans, Pimonidazole, Carbonic Anhydrase IX, Transcription factor, Carbonic Anhydrases, Tumor hypoxia, Hypoxia (medical), Tumor Oxygenation, Flow Cytometry, Hypoxia-Inducible Factor 1, alpha Subunit, Cell Hypoxia, Transplantation, Oncology, Nitroimidazoles, Cell culture, Colonic Neoplasms, Cancer research, Female, medicine.symptom, Transcription Factors
Abstract

Hypoxia that develops in solid tumors stabilizes the hypoxia-inducible factor-1α (HIF-1α) subunit of the HIF-1 transcription factor, leading to up-regulation of dozens of hypoxia-regulated genes that increase glycolysis and oxygen delivery. HIF-1α and its downstream target gene CA9 have both been used as surrogate hypoxia markers, and, in general, high expression predicts for a poor response to treatment. Combinations of hypoxia markers offer the opportunity to measure changes in tumor oxygenation that may be relevant to tumor response to treatment. We compared the degree of colocalization of two endogenous markers for hypoxia, HIF-1α and carbonic anhydrase IX (CAIX), with a chemical marker for hypoxia, pimonidazole. Unexpectedly, expression of HIF-1α was reduced in the most hypoxic regions that border necrosis in xenograft tumors composed of SiHa cervical carcinoma, WiDr colon carcinoma, or M006 astrocytoma cells. Similar results were obtained for samples from three cervical cancer biopsies. However, CAIX was present in these perinecrotic cells that were also capable of metabolizing and binding a chemical marker for hypoxia, pimonidazole. In vitro experiments using tumor cells and tumor cubes incubated under anoxic conditions indicated that nutrient deprivation seems to be largely responsible for the lack of HIF-1α expression in perinecrotic regions. The half-life of CAIX was sufficiently long that, once formed, it remained for days in the absence of continued HIF-1α expression. These results have implications for the use of HIF-1α as an indicator of tumor hypoxia and aggressiveness as well as development of hypoxia-directed antitumor therapies based on the expression of HIF-1α.

Published
2005

13. BlaR1 and MecR1 Gene Products of Staphylococcus aureus

Author

Natalie C. J. Strynadka, Solmaz Sobhanifar, and Gerd Prehna

Subjects
Transposable element, Genetics, Biology, biology.organism_classification, medicine.disease_cause, Transmembrane protein, Microbiology, Serine, Plasmid, Staphylococcus aureus, Gene cluster, medicine, Bacillus licheniformis, Integral membrane protein, Gene
Abstract

Publisher Summary This chapter discusses the structural chemistry and the biological aspects of BlaR1 and MecR1 gene products of Staphylococcus aureus. The gene cluster encoding regulated production of β-lactamase in Staphylococcus aureus is usually found on a plasmid or a transposon. The cluster includes a repressor gene bla1, an anti-repressor gene blaR1 and the β-lactamase gene blaZ. While the protease activity of BlaR1 is yet to be demonstrated in vitro, the absolute requirement for the intact HEXXH zinc-metalloprotease motif in cleavage of Bla1 suggests that activated BlaR1 probably cleaves Bla1 in vivo. BlaR1 and MecR1 show amino acid homology, and an overall structural similarity to PenR1 of Bacillus licheniformis. Based on detailed structural studies on PenR1, BlaR1 and MecR1 are predicted to be integral membrane proteins with four α-helical transmembrane segments. The overall structure of the protein suggests that it is a natural fusion of a penicillin-binding protein to a metalloprotease. C-terminal large extracellular domain encodes a penicilloyl serine transferase sensor that contains the STYK, YGN and KTG motifs conserved among all penicillin-binding proteins.

Published
2013

14. Contributors

Author

Catherine Anne Abbott, Carmela R. Abraham, Hideki Adachi, Osao Adachi, Zach Adam, Michael W.W. Adams, Michael J. Adang, Ibrahim M. Adham, Patrizia Aducci, David A. Agard, Alexey A. Agranovsky, Tetsuya Akamatsu, Yoshinori Akiyama, Reidar Albrechtsen, Alí Alejo, Sean M. Amberg, Alexander Y. Amerik, Piti Amparyup, Felipe Andrade, Germán Andrés, Daniel M. Andrews, Robert K. Andrews, Toni M. Antalis, Colin S. Anthony, Naoya Aoki, Suneel S. Apte, Kazunari Arima, Gérard Arlaud, Raghuvir Krishnaswamy Arni, Pascal Arnoux, Nathan N. Aronson, Michel Arthur, Yasuhisa Asano, Paolo Ascenzi, Marina T. Assakura, David S. Auld, Veridiana de Melo Rodrigues Ávila, Francesc X. Avilés, William M. Awad, Anand K. Bachhawat, Shan Bai, Teaster T. Baird, S. Paul Bajaj, Susan C. Baker, Agnieszka Banbula, Alan J. Barrett, Jemima Barrowman, John D. Bartlett, Jörg W. Bartsch, Nikola Baschuk, Isolda P. Baskova, Jyotsna Batra, Karl Bauer, Ulrich Baumann, Wolfgang Baumeister, Cédric Bauvois, Alex Bayés, Anne Beauvais, Christoph Becker-Pauly, Tadhg P. Begley, Miklós Békés, Robert Belas, Daniah Beleford, Teruhiko Beppu, Ernst M. Bergmann, Bruno A. Bernard, Dominique Bernard, Michael C. Berndt, Giovanna Berruti, Colin Berry, Greg P. Bertenshaw, Christian Betzel, Chetana Bhaskarla, Manoj Bhosale, Gabriele Bierbaum, B. Bjarnason Jón, Michael Blaber, Michael J. Blackman, Alexander Blinkovsky, Jef D. Boeke, Matthew Bogyo, Stefan Bohn, Guy Boileau, Mike Boland, Tové C. Bolken, Judith S. Bond, Jan Bondeson, Javier Bordallo, Claudia Borelli, Tiago O. Botelho, Richard R. Bott, David G. Bourne, Niels Bovenschen, Ralph A. Bradshaw, Klaus Breddam, Keith Brew, Paul J. Brindley, Diane L. Brinkman, Collette Britton, Jeff R. Broadbent, Anne Broadhurst, Dieter Brómme, Murray Broom, Jeremy S. Brown, Mark A. Brown, Iris Bruchhaus, Barbara A. Burleigh, Kristin E. Burns, James F. Burrows, Michael J. Butler, David J. Buttle, Chelsea M. Byrd, Tony Byun, Sandrine Cadel, Conor R. Caffrey, Santiago Cal, Javier Caldentey, Thomas Candela, Clemente Capasso, Daniel R. Capriogilio, Vincenzo Carginale, Adriana Karaoglanovic Carmona, Vern B. Carruthers, Francis J. Castellino, Joseph J. Catanese, Bruce Caterson, George H. Caughey, Naimh X. Cawley, Tim E. Cawston, Juan José Cazzulo, Jijie Chai, Karl X. Chai, Olga Meiri Chaim, L.S. Chang, Julie Chao, Marie-Pierre Chapot-Chartier, Jean-Louis Charli, Paulette Charlier, Karen J. Chave, Jian-Min Chen, Jinq-May Chen, Li-Mei Chen, Ya-Wen Chen, Yu-Yen Chen, Bernard Chevrier, Jean-François Chich, Jeremy Chien, Suneeta Chimalapati, Ki Joon Cho, Kwan Yong Choi, Woei-Jer Chuang, Chin Ha Chung, Ivy Yeuk Wah Chung, Christine Clamagirand, Ian M. Clark, Adrian K. Clarke, Nicola E. Clarke, Steven Gerard Clarke, Philippe Clauziat, Judith A. Clements, Catherine Coffinier, Paul Cohen, Alain Colige, Anne Collignon, Sean D. Colloms, Andreas Conzelmann, Graham H. Coombs, Jakki C. Cooney, Jonathan B. Cooper, Max D. Cooper, Nikki A. Copeland, Graeme S. Cottrell, Joseph T. Coyle, Charles S. Craik, John W.M. Creemers, Daniela Cretu, Jenifer Croce, Keith J. Cross, Rosario Cueva, Sheng Cui, Luis Cunha, Simon Cutting, Christophe d’Enfert, Hugues D’Orchymont, Björn Dahlbäck, Shujia Dai, Ross E. Dalbey, John P. Dalton, Pam M. Dando, R.M. Daniel, Sergei M. Danilov, Donna E. Davies, Heloisa S. De Araujo, Teresa De los Santos, Viviana de Luca, Ingrid De Meester, Ana Karina de Oliveira, Eduardo Brandt de Oliveira, Pedro Lagerblad De Oliveira, Sarah de Vos, Jeroen Declercq, Wim Declercq, Ala-Eddine Deghmane, Niek Dekker, Sonia Del Prete, Marina Del Rosal, Bernard Delmas, Robert DeLotto, Ilya V. Demidyuk, Mark R. Denison, Jan M. Deussing, Lakshmi A. Devi, Eleftherios P. Diamandis, Isabel Diaz, Araceli Díaz-Perales, Bauke W. Dijkstra, Yan Ding, Jack E. Dixon, Johannes Dodt, Terje Dokland, Iztok Dolenc, Ningzheng Dong, Tran Cat Dong, Ying Dong, Mitesh Dongre, Mark Donovan, Timothy M. Dore, Loretta Dorstyn, Hong Dou, Zhicheng Dou, Annette M. Dougall, Marcin Drag, Edward G. Dudley, Ben M. Dunn, Bruno Dupuy, Maria Conceicāo Duque-Magalhāes, M. Asunción Durá, Yves Eeckhout, Vincent Eijsink, Arthur Z. Eisen, Azza Eissa, Sandra Eklund, Ziad M. Eletr, Vincent Ellis, Wolfgang Engel, Ervin G. Erdös, Teresa Escalante, David A. Estell, Michael Etscheid, Herbert J. Evans, Roger D. Everett, Alex C. Faesen, Falk Fahrenholz, Miriam Fanjul-Fernández, Christopher J. Farady, Georges Feller, Hong Feng, Kurt M. Fenster, Claude Férec, Silvia Ferrari, Barbara Fingleton, Jed F. Fisher, Paula M. Fives-Taylor, Loren G. Fong, F. Forneris, Brian M. Forster, Friedrich Forster, Simon J. Foster, Thierry Foulon, Stephen I. Foundling, Jay William Fox, Bruno Franzetti, Alejandra P. Frasch, Hudson H. Freeze, Jean-Marie Frère, Teryl K. Frey, Beate Fricke, Lloyd D. Fricker, Rafael Fridman, Christopher J. Froelich, Camilla Fröhlich, Hsueh-Liang Fu, Cynthia N. Fuhrmann, Satoshi Fujimura, Hiroshi Fujiwara, Jun Fukushima, Keiichi Fukuyama, Robert S. Fuller, Martin Fusek, Christine Gaboriaud, Christian Gache, Oleksandr Gakh, Peter Gal, Junjun Gao, Adolfo García-Sastre, Donald L. Gardiner, John A. Gatehouse, G.M. Gaucher, Francis Gauthier, Jean-Marie Ghuysen, Wade Gibson, Jennifer Gillies, Elzbieta Glaser, Fabian Glaser, Michael H. Glickman, Peter Goettig, Colette Goffin, Eiichi Gohda, Alfred L. Goldberg, Daniel E. Goldberg, Gregory I. Goldberg, Nathan E. Goldfarb, F. Xavier Gomis-Rüth, B. Gopal, Alexander E. Gorbalenya, Stuart G. Gordon, Mark D. Gorrell, Friedrich Götz, Theodoros Goulas, Cécile Gouzy-Darmon, K. Govind, Lászlo Gráf, Robert R. Granados, Melissa Ann Gräwert, Douglas A. Gray, Thomas P. Graycar, Jonathan A. Green, Luiza Helena Gremski, Michael Groll, Tania Yu Gromova, P. Gros, Marvin J. Grubman, Amy M. Grunden, Ágústa Gudmundsdóttir, Micheline Guinand, Djamel Gully, Alla Gustchina, José María Gutiérrez, Byung Hak Ha, Jesper Z. Haeggström, James H. Hageman, Johanna Haiko, Stephan Hailfinger, Hans Michael Haitchi, Ji Seon Han, Chantal Hanquez, Minoru Harada, Ikuko Hara-Nishimura, Marianne Harboe, Torleif Härd, David A. Harris, Ulrich Hassiepen, Shoji Hata, Akira Hattori, Rong-Qiao He, Albert J.R. Heck, Dirk F. Hendricks, Bernhard Henrich, Patrick Henriet, Andrés Hernández-Arana, Irma Herrera-Camacho, Gerhard Heussipp, Toshihiko Hibino, P.M. Hicks, Bradley I. Hillman, B. Yukihiro Hiraoka, Jun Hiratake, Yohei Hizukuri, Heng-Chien Ho, Ngo Thi Hoa, Mark Hochstrasser, Kathryn M. Hodge, Theo Hofmann, Thomas Hohn, John R. Hoidal, Joachim-Volker Höltje, Koichi J. Homma, John F. Honek, Vivian Y.H. Hook, John D. Hooper, Nigel M. Hooper, Kazuo Hosoi, Christopher J. Howe, Dennis E. Hruby, James J.-D. Hseih, Chun-Chieh Hsu, Tony T. Huang, Tur-Fu Huang, Yoann Huet, Clare Hughes, Jean-Emmanuel Hugonnet, Adrienne L. Huston, Oumaïma Ibrahim-Granet, Eiji Ichishima, Yukio Ikehara, Tadashi Inagami, Jessica Ingram, R.E. Isaac, Grazia Isaya, Clara E. Isaza, Shin-ichi Ishii, Amandine Isnard, Kiyoshi Ito, Koreaki Ito, Yoshifumi Itoh, Xavier Iturrioz, Sadaaki Iwanaga, Ralph W. Jack, Mel C. Jackson, Michael N.G. James, Jiří Janata, Claire Janoir, Hanna Janska, Ken F. Jarrell, Mariusz Jaskolski, Sheila S. Jaswal, Ying Y. Jean, Dieter E. Jenne, Young Joo Jeon, Ping Jiang, John E. Johnson, Michael D. Johnson, James A. Johnston, Amanda Jones, Elizabeth W. Jones, Carine Joudiou, Luiz Juliano, Hea-Jin Jung, Ray Jupp, Todd F. Kagawa, Hubert Kalbacher, Yayoi Kamata, Shuichi Kaminogawa, Yoshiyuki Kamio, Makoto Kaneda, Sung Gyun Kang, Sung Hwan Kang, Mary Kania, Tomasz Kantyka, Nobuyuki Kanzawa, Abdulkarim Y. Karim, Takafumi Kasumi, Hiroaki Kataoka, Hardeep Kaur, Shun-Ichiro Kawabata, Mari Kawaguchi, John Kay, Murat Kaynar, Kenneth C. Keiler, R.M. Kelly, Nathaniel T. Kenton, Michael A. Kerr, Kristof Kersse, Jukka Kervinen, Benedikt M. Kessler, Efrat Kessler, Timo K. Khoronen, Simon Kidd, Marjolein Kikkert, Mogens Kilian, Do-Hyung Kim, Doyoun Kim, Eunice EunKyeong Kim, In Seop Kim, Jung-Gun Kim, Kyeong Kyu Kim, Kyung Hyun Kim, Matthew S. Kimber, Yukio Kimura, Heidrun Kirschke, Yoshiaki Kiso, Colin Kleanthous, Jürgen R. Klein, Michael Klemba, Beata Kmiec, Hideyuki Kobayashi, Hiroyuki Kodama, Gerald Koelsch, Jan Kok, P.E. Kolattukody, Fabrice A. Kolb, Harald Kolmar, Yumiko Komori, Jan Konvalinka, Brice Korkmaz, Sergey V. Kostrov, Hans-Georg Kräusslich, Gabi Krczal, Lawrence F. Kress, Magnüs Már Kristjánsson, Tomáš Kučera, Sayali S. Kukday, Hidehiko Kumagai, Sharad Kumar, Malika Kumarasiri, Takashi Kumazaki, Beate M. Kümmerer, Kouji Kuno, Markku Kurkinen, Eva Kutejová, Marie Kveiborg, Agnieszka Kwarciak, Liisa Laakkonen, Nikolaos E. Labrou, Gavin D. Laing, Gayle Lamppa, Thomas Langer, Richard A. Laursen, Richard A. Lawrenson, Matthew D. Layne, Bernard F. Le Bonniec, María C. Leal, Ronald M. Lechan, David H. Lee, Irene Lee, Jae Lee, Kye Joon Lee, Soohee Lee, Xiaobo Lei, Jonathan Leis, Ellen K. LeMosy, Thierry Lepage, Stephen H. Leppla, Adam Lesner, Ivan A.D. Lessard, Guy Lhomond, Huilin Li, Shu-Ming Li, Weiguo Li, Ta-Hsiu Liao, Robert C. Liddington, Toby Lieber, H.R. Lijnen, Christopher D. Lima, Chen-Yong Lin, Gang Lin, Ming T. Lin, Xinli Lin, Yee-Shin Lin, L.L. Lindsay, William N. Lipscomb, John W. Little, Ching-Chuan Liu, Chuan-ju Liu, Mark O. Lively, Nurit Livnat-Levanon, Per O. Ljungdahl, Catherine Llorens-Cortes, Peter Lobel, Y. Peng Loh, Jouko Lohi, G.P. Lomonossoff, Yvan Looze, Carlos López-Otin, Landys Lopez-Quezada, Alex Loukas, Long-Sheng Lu, Áke Lundwall, Liu-Ying Luo, Andrei Lupas, Dawn S. Luthe, Nicholas J. Lynch, Peter J. Lyons, Vivian L. MacKay, Jesica M. Levingston Macleod, Viktor Magdolen, Jean-Luc Mainardi, Kauko K. Mäkinen, Jeremy P. Mallari, Surya P. Manandhar, Fajga R. Mandelbaum, Anne M. Manicone, Johanna Mansfeld, Joseph Marcotrigiano, Michael Mares, Gemma Marfany, Francis S. Markland, Judith Marokházi, Hélène Marquis, Robert A. Marr, Enzo Martegani, Erik W. Martin, Manuel Martinez, L. Miguel Martins, Masato Maruyama, Masugi Maruyama, Sususmu Maruyama, Takeharu Masaki, Ramin Massoumi, Rency T. Mathew, Lynn M. Matrisian, Yoshihiro Matsuda, Osamu Matsushita, Marco Matuschek, Anna Matušková, Krisztina Matúz, Cornelia Mauch, Michael R. Maurizi, Lorenz M. Mayr, Dewey G. McCafferty, J. Ken McDonald, James H. McKerrow, David McMillan, Robert P. Mecham, Darshini P. Mehta, Chris Meisinger, Alan Mellors, Roger G. Melton, Jeffrey A. Melvin, Robert Ménard, Luis Menéndez-Arias, Milene C. Menezes, Andrew Mesecar, Stéphane Mesnage, Diane H. Meyer, Gregor Meyers, Susan Michaelis, Karolina Michalska, Wojciech P. Mielicki, Igor Mierau, Galina V. Mikoulinskaia, Charles G. Miller, Lydia K. Miller, John Mills, Kenneth V. Mills, Jinrong Min, Michel-Yves Mistou, Yoshio Misumi, Shin-ichi Miyoshi, Shigehiko Mizutani, Shahriar Mobashery, Satsuki Mochizuki, William L. Mock, Frank Möhrlen, Nathalie Moiré, Paul E. Monahan, Angela Moncada-Pazos, Véronique Monnet, Michel Monod, Cesare Montecucco, Laura Morelli, Sumiko Mori, Takashi Morita, James H. Morrissey, Richard J. Morse, John S. Mort, Uffe H. Mortensen, Rory E. Morty, Joel Moss, Hidemasa Motoshima, Jeremy C. Mottram, Ana M. Moura-da-Silva, Mary Beth Mudgett, Egbert Mundt, Kazuo Murakami, Mario Tyago Murakami, Kimiko MurakamiMurofoshi, Sawao Murao, Gillian Murphy, M.R.N. Murthy, Tatsushi Muta, Elmarie Myburgh, Nino Mzhavia, A.H.M. Nurun Nabi, Hideaki Nagase, Michael W. Nagle, Dorit K. Nägler, Rajesh R. Naik, Divya B. Nair, Toshiki Nakai, Yoshitaka Nakajima, Yukio Nakamura, Hitoshi Nakatogawa, Toru Nakayama, Natalia N. Nalivaeva, Dipankar Nandi, Maria Clara Leal Nascimento-Silva, Kim Nasmyth, Carl F. Nathan, Fernando Navarro-García, Dayane Lorena Naves, Danny D. Nedialkova, Keir C. Neuman, Jeffrey-Tri Nguyen, Ky-Anh Nguyen, Gabriela T. Niemirowicz, Toshiaki Nikai, Eiichiro Nishi, Wataru Nishii, Makoto Nishiyama, Yasuhiro Nishiyama, Masatoshi Noda, Seiji Nomura, Shigemi Norioka, Desire M.M. Nsangou, Amornrat O’Brien, Michael B. O’Connor, Kohei Oda, Irina V. Odinokova, Joyce Oetjen, Teru Ogura, Dennis E Ohman, Yoshinori Ohsumi, Mukti Ojha, Akinobu Okabe, Yasunori Okada, Keinosuke Okamoto, Kenji Okuda, Nobuaki Okumura, Takashi Okuno, Kjeld Oleson, Priscila Oliveira de Giuseppe, Martin Olivier, Yasuko Ono, Stephen Oroszlan, Nobuyuki Ota, Michael Ovadia, Jiyang O-Wang, Claus Oxvig, Jeremy C.L. Packer, Sergio Padilla-López, Mark Paetzel, Michael J. Page, Andrea Page-McCaw, Mark J.I. Paine, Byoung Chul Park, Eunyong Park, John E. Park, Pyong Woo Park, Sung Goo Park, Kirk L. Parkin, William C Parks, Thaysa Paschoalin, Annalisa Pastore, Alexander Nikolich Patananan, Sudhir Paul, Henry L. Paulson, Ulrich von Pawel-Rammingen, David A. Pearce, Mark S. Pearson, Duanqing Pei, Gunnar Pejler, Alan D. Pemberton, Jianhao Peng, Julien Pernier, Jan-Michael Peters, Thorsten Pfirrmann, Viet-Laï Pham, Iva Pichová, Darren Pickering, Christophe Piesse, David Pignol, Robert N. Pike, Lothaire Pinck, Hubert Pirkle, Henry C. Pitot, Andrew G. Plaut, Hidde Ploegh, László Polgár, Corrine Porter, Rolf Postina, Jan Potempa, Knud Poulsen, Scott D. Power, Rex. F. Pratt, Gerd Prehna, Gilles Prévost, Alexey V. Pshezhetsky, Mohammad A. Qasim, Feng Qian, Jiazhou Qiu, Víctor Quesada, Evette S. Radisky, Stephen D. Rader, Kavita Raman, Andrew J. Ramsay, Derrick E. Rancourt, Najju Ranjit, Narayanam V. Rao, Kiira Ratia, Neil D. Rawlings, Robert B. Rawson, Vijay Reddy, Colvin M. Redman, Maria Elena Regonesi, Andreas S. Reichert, Antonia P. Reichl, Han Remaut, S. James Remington, Martin Renatus, David Reverter, Eric C. Reynolds, Mohamed Rholam, Charles M. Rice, Todd W. Ridky, Howard Riezman, D.C. Rijken, Marie-Christine Rio, Alison Ritchie, Janine Robert-Baudouy, Mark W. Robinson, Michael Robinson, Adela Rodriguez-Romero, Renata Santos Rodriques, John C. Rogers, Camilo Rojas, Floyd E. Romesberg, David J. Roper, Nora Rosas-Murrieta, A.M. Rose, Philip J. Rosenthal, J. Rosing, Ornella Rossetto, Véronique Rossi, Richard A. Roth, Hanspeter Rottensteiner, Andrew D. Rowan, Mikhail Rozanov, Alexandra Rucavado, Andrea Ruecker, Françoise Rul, Till Rümenapf, Ilaria Russo, Martin D. Ryan, Elena Sacco, J. Evan Sadler, W. Saenger, Hans-Georg Sahl, Mohammed Sajid, Masayoshi Sakaguchi, Fumio Sakiyama, Maria L. Salas, Maria Cristina O. Salgado, Guy S. Salvesen, Edith Sánchez, Eladio F. Sanchez, Qing-Xiang Amy Sang, Krishnan Sankaran, Susanta K. Sarkar, Michael P. Sarras, Yoshikiyo Sasagawa, Araki Satohiko, Eric Sauvage, Loredana Saveanu, H.S. Savithri, Hitoshi Sawada, R. Gary Sawers, Isobel A. Scarisbrick, Andreas Schaller, Justin M. Scheer, Friedrich Scheiflinger, Cordelia Schiene-Fischer, Uwe Schlomann, Manfred Schlösser, Alvin H. Schmaier, Walter K. Schmidt, Anette Schneemann, Rick G. Schnellmann, Henning Scholze, Lutz Schomburg, Wilhelm J. Schwaeble, Christopher J. Scott, Rosaria Scudiero, Atsuko Sehara-Fujisawa, Nabil G. Seidah, Motoharu Seiki, Junichi Sekiguchi, Andrea Senff-Ribeiro, Ihn Sik Seong, Mihaela Serpe, Solange M.T. Serrano, Peter Setlow, Tina Shahian, M. Shanks, Feng Shao, Steven D. Shapiro, Navneet Sharma, Lindsey N. Shaw, Aimee Shen, Lei Shen, Roger F. Sherwood, Yun-Bo Shi, Hitoshi Shimoi, Yoichiro Shimura, A.D. Shirras, Viji Shridhar, Jinal K. Shukla, Ene Siigur, Jüri Siigur, Natalie C. Silmon de Monerri, Robert B. Sim, James P. Simmer, William H. Simmons, Jaspreet Singh, Alison Singleton, Tatiana D. Sirakova, Titia K. Sixma, Tim Skern, Randal A. Skidgel, Jeffrey Slack, David E. Sleat, Barbara S. Slusher, Janet L. Smith, Matthew A. Smith, Mark J. Smyth, Erik J. Snijder, Solmaz Sobhanifar, Kenneth Söderhaäll, Istvan Sohar, Peter Sonderegger, Marcos Henrique Ferreira Sorgine, Hiroyuki Sorimachi, Karen E. Soukhodolets, Tatiana de Arruda Campos Brasil de Souza, Tamás Sperka, Shiranee Sriskandan, Joseph W. St. Geme, Raymond J. St. Leger, Peter Staib, James L. Steele, Bjarki Stefansson, Christian Steinkühler, Leisa M. Stenberg, Johan Stenflo, Henning R. Stennicke, Valentin M. Stepanov, Olga A. Stepnaya, Frank Steven, Richard L. Stevens, Kenneth J. Stevenson, Mathieu St-Louis, Christopher C. Stobart, Walter Stöcker, Andrew C. Storer, Norbert Sträter, Ellen G. Strauss, James H. Strauss, Kvido Stříšovský, Natalie C.J. Strynadka, Edward D. Sturrock, Dan Su, Xiao-Dong Su, Paz Suárez-Rendueles, Traian Sulea, Venkatesh Sundararajan, Ryoji Suno, Carolyn K. Suzuki, Fumiaki Suzuki, Hideyuki Suzuki, Nobuhiro Suzuki, Stephen Swenson, Rose L. Szabady, Pal Bela Szecsi, Lászlo Szilágyi, Muhamed-Kheir Taha, Eizo Takahashi, Kenji Takahashi, Toshiro Takai, Atsushi Takeda, Soichi Takeda, Jeremy J.R.H. Tame, Tomohiro Tamura, Fulong Tan, Keiji Tanaka, Carmen Tanase, Jordan Tang, Martha M. Tanizaki, Egbert Tannich, Guido Tans, Anthony L. Tarentino, Anchalee Tassanakajon, Hiroki Tatsumi, Norbert Tautz, Erin Bassford Taylor, Pedro Filipe Teixeira, Bhanu Prakash V.L. Telugu, Markus F. Templin, Shigeyuki Terada, Uchikoba Tetsuya, C. Thacker, Maulik Thaker, Heinz-Jürgen Thiel, Nicole Thielens, Gonzales Thierry, Karine Thivierge, Mark D. Thomas, Margot Thome, Mary K. Thorsness, Peter E. Thorsness, Natalie J. Tigue, Sokol V. Todi, Birgitta Tomkinson, Fiorella Tonello, Liang Tong, H.S. Toogood, Paolo Tortora, József Tözsèr, Luiz Rodolpho Travassos, James Travis, Dilza Trevisan-Silva, Francesca Trinchella, Neil N. Trivedi, Carol M. Troy, Harald Tschesche, Yu-Lun Tseng, Masafumi Tsujimoto, Anthony T. Tu, Kathleen E. Tumelty, Boris Turk, Dusan Turk, Vito Turk, Anthony J. Turner, Tetsuya Uchikoba, Takayuki Ueno, Alejandro P. Ugalde, Veli-Jukka Uitto, Sinisa Urban, Olivier Valdenaire, Adrian Valli, Jozef Van Beeumen, Bertus Van den Burg, Renier A.L. Van der Hoorn, Jan Maarten van Dijl, Peter Van Endert, Bram J. Van Raam, Harold E. Van Wart, Tom Vanden Berghe, Peter Vandenabeele, Margo Vanoni, Silvio Sanches Veiga, William H. Velander, Gloria Velasco, Josep Vendrell, I. István Venekei, Vaclav Vetvicka, F.-Nora Vögtle, Waldemar Vollmer, Kei Wada, Fred W. Wagner, Sun Nyunt Wai, Timothy Wai, Shane Wainwright, Kenneth W. Walker, Stephen J. Walker, Jean Wallach, Linda L. Walling, Peter N. Walsh, Hai-Yan Wang, Hengbin Wang, Jianwei Wang, Peng Wang, Ping Wang, Michael Wassenegger, Kunihiko Watanabe, Helen Webb, Joseph M. Weber, Niklas Weber, Daniel R. Webster, Shuo Wei, Rodney A. Welch, James A. Wells, Herbert Wenzel, Ingrid E. Wertz, Ulla W. Wewer, Alison R. Whyteside, Sherwin Wilk, Jean-Marc Wilkin, Claudia Wilmes, Jakob R. Winther, David S. Wishart, Alexander Wlodawer, J. Fred Woessner, Michael S. Wolfe, Wilson Wong, Roger Woodgate, Gerry Wright, Jiunn-Jong Wu, Qingyu Wu, Magdalena Wysocka, Chao Xu, Zhenghong Xu, Kinnosuke Yahori, Shoji Yamada, Nozomi Yamaguchi, Shinji Yamaguchi, Yoshio Yamakawa, Hiroki Yamamoto, Ikao Yana, Maozhou Yang, Na Yang, Chenjuan Yao, Tingting Yao, Noriko Yasuda, Toshimasa Yasuhara, Shigeki Yasumasu, Edward T.H. Yeh, Irene Yiallouros, Jiang Yin, Hiroo Yonezawa, Soon Ji Yoo, Tadashi Yoshimoto, Michael W. Young, Stephen G. Young, Nousheen Zaidi, Ludmila L. Zavalova, Peter Zavodszky, Aidong Zhang, Xianming Zhang, Yi-Zheng Zhang, Dominick Zheng, Guangming Zhong, Rong Zhong, Yuan Zhou, Zhaohui Sunny Zhou, Michael Zick, Paola Zigrino, and Andrei A. Zimin

Published
2013

15. A method for integrative structure determination of protein-protein complexes

Author

Hong Liang, Jaume Pons, Ho Min Kim, Dina Schneidman-Duhovny, Pavel Strop, Kristin A. Krukenberg, Volker Dötsch, Seung Joong Kim, Maofu Liao, David A. Agard, Yifan Cheng, Andrej Sali, Andrea Rossi, Agustin Avila-Sakar, Javier Velázquez-Muriel, Solmaz Sobhanifar, and Arvind Rajpal

Subjects
Statistics and Probability, Small Angle, Materials science, Bioinformatics, 1.1 Normal biological development and functioning, Bioengineering, Antigen-Antibody Complex, Mass spectrometry, Crystallography, X-Ray, Biochemistry, Electron, Mathematical Sciences, Protein–protein interaction, Scattering, X-Ray Diffraction, Underpinning research, Information and Computing Sciences, Microscopy, Scattering, Small Angle, Molecular Biology, Crystallography, Small-angle X-ray scattering, Protein protein, Experimental data, Nuclear magnetic resonance spectroscopy, Biological Sciences, Original Papers, Computer Science Applications, Molecular Docking Simulation, Computational Mathematics, Microscopy, Electron, Computational Theory and Mathematics, Networking and Information Technology R&D (NITRD), Docking (molecular), Multiprotein Complexes, X-Ray, Generic health relevance, Biological system, Software
Abstract

Motivation: Structural characterization of protein interactions is necessary for understanding and modulating biological processes. On one hand, X-ray crystallography or NMR spectroscopy provide atomic resolution structures but the data collection process is typically long and the success rate is low. On the other hand, computational methods for modeling assembly structures from individual components frequently suffer from high false-positive rate, rarely resulting in a unique solution. Results: Here, we present a combined approach that computationally integrates data from a variety of fast and accessible experimental techniques for rapid and accurate structure determination of protein–protein complexes. The integrative method uses atomistic models of two interacting proteins and one or more datasets from five accessible experimental techniques: a small-angle X-ray scattering (SAXS) profile, 2D class average images from negative-stain electron microscopy micrographs (EM), a 3D density map from single-particle negative-stain EM, residue type content of the protein–protein interface from NMR spectroscopy and chemical cross-linking detected by mass spectrometry. The method is tested on a docking benchmark consisting of 176 known complex structures and simulated experimental data. The near-native model is the top scoring one for up to 61% of benchmark cases depending on the included experimental datasets; in comparison to 10% for standard computational docking. We also collected SAXS, 2D class average images and 3D density map from negative-stain EM to model the PCSK9 antigen–J16 Fab antibody complex, followed by validation of the model by a subsequently available X-ray crystallographic structure. Availability: http://salilab.org/idock Contact: dina@salilab.org or sali@salilab.org Supplementary information: Supplementary data are available at Bioinformatics online.

Published
2012

16. Structural Investigation of Cell-Free Expressed Membrane Proteins

Author

Frank Bernhard, Sina Reckel, Frank Löhr, Solmaz Sobhanifar, and Volker Dötsch

Subjects
Vesicle-associated membrane protein 8, biology, Membrane protein, Membrane transport protein, Chemistry, Peripheral membrane protein, biology.protein, Cell free, Integral membrane protein, Cell biology, Transport protein
Published
2012

17. Strategies for the cell-free expression of membrane proteins

Author

Sina, Reckel, Solmaz, Sobhanifar, Florian, Durst, Frank, Löhr, Vladimir A, Shirokov, Volker, Dötsch, and Frank, Bernhard

Subjects
Protein Folding, Cell-Free System, Transcription, Genetic, Protein Conformation, Detergents, Membrane Proteins, Protein Engineering, Recombinant Proteins, Membrane Lipids, Protein Biosynthesis, Escherichia coli, Animals, Humans, RNA, Messenger, Hydrophobic and Hydrophilic Interactions, Nuclear Magnetic Resonance, Biomolecular
Abstract

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

Published
2010

18. Strategies for the Cell-Free Expression of Membrane Proteins

Author

Florian Durst, Solmaz Sobhanifar, Volker Dötsch, Sina Reckel, V.A. Shirokov, Frank Löhr, and Frank Bernhard

Subjects
Folding (chemistry), Membrane protein, biology, Chemistry, biology.protein, Biophysics, Cell free, Expression (computer science), Lipid bilayer, Integral membrane protein, Micelle, Cofactor
Abstract

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

Published
2009

19. Cell-free expression and stable isotope labelling strategies for membrane proteins

Author

Solmaz Sobhanifar, Mikhail Karbyshev, Lei Kai, Sina Reckel, Volker Dötsch, Frank Löhr, Frank Bernhard, Friederike Junge, and Daniel Schwarz

Subjects
chemistry.chemical_classification, Cell-Free System, Protein Conformation, Cell, Membrane Proteins, Computational biology, Biochemistry, Transmembrane protein, Inclusion bodies, Amino acid, medicine.anatomical_structure, Protein structure, Membrane, chemistry, Membrane protein, Labelling, Isotope Labeling, medicine, Nuclear Magnetic Resonance, Biomolecular, Spectroscopy
Abstract

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on alpha-helical transmembrane proteins which benefit most from such methods.

Published
2009

20. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

Author

Frank Bernhard, Volker Dötsch, Friederike Junge, Birgit Schneider, Nadine Frölich, Sina Reckel, Solmaz Sobhanifar, Florian Durst, and Daniel Schwarz

Subjects
Cell Extracts, Cell-Free System, Detergents, Membrane Proteins, Protein engineering, Biology, medicine.disease_cause, Proteomics, Lipid Metabolism, General Biochemistry, Genetics and Molecular Biology, chemistry.chemical_compound, Structural biology, Membrane protein, Biochemistry, chemistry, medicine, Escherichia coli, T7 RNA polymerase, DNA microarray, DNA, medicine.drug
Abstract

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

Published
2007

21. Investigation of potential drug targets involved in antibiotic sensing and cell wall modification

Author

Gregory A. Wasney, Robert J. Gruninger, Solmaz Sobhanifar, Liam J. Worrall, and Natalie C. J. Strynadka

Subjects
Drug, medicine.drug_class, Chemistry, media_common.quotation_subject, Antibiotics, Pharmacology, Condensed Matter Physics, Biochemistry, Inorganic Chemistry, Structural Biology, medicine, General Materials Science, Physical and Theoretical Chemistry, Cell wall modification, media_common
Abstract

The emergence of an increasing number of broad-spectrum antibiotic resistant bacteria such as MRSA over the last decade has made the understanding of the mechanisms underlying resistance critical. Resistance very often involves enzymes associated with the cell wall, a common target of antibiotics. Here we investigate the structure and function of enzymes involved in antibiotic sensing and cell wall modification, while using a customized "cell-free" protein synthesis approach to obtain sufficient yields of promising but difficult-to-express antibacterial drug targets.

Published
2014

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