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2. Aetiology of Type 2 diabetes in people with a ‘normal’ body mass index: testing the personal fat threshold hypothesis

Author

Taylor, Roy, primary, Barnes, Alison C., additional, Hollingsworth, Kieren G., additional, Irvine, Keaton M., additional, Solovyova, Alexandra S., additional, Clark, Lucy, additional, Kelly, Tara, additional, Martin-Ruiz, Carmen, additional, Romeres, Davide, additional, Koulman, Albert, additional, Meek, Claire M., additional, Jenkins, Benjamin, additional, Cobelli, Claudio, additional, and Holman, Rury R., additional

Published
2023
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9. Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli

Author

Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Banzhaf, Manuel, Yau, Hamish Cl, Verheul, Jolanda, Lodge, Adam, Kritikos, George, Mateus, André, Cordier, Baptiste, Hov, Ann Kristin, Stein, Frank, Wartel, Morgane, Pazos, Manuel, Solovyova, Alexandra S, Breukink, Eefjan, van Teeffelen, Sven, Savitski, Mikhail M, den Blaauwen, Tanneke, Typas, Athanasios, Vollmer, Waldemar, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Banzhaf, Manuel, Yau, Hamish Cl, Verheul, Jolanda, Lodge, Adam, Kritikos, George, Mateus, André, Cordier, Baptiste, Hov, Ann Kristin, Stein, Frank, Wartel, Morgane, Pazos, Manuel, Solovyova, Alexandra S, Breukink, Eefjan, van Teeffelen, Sven, Savitski, Mikhail M, den Blaauwen, Tanneke, Typas, Athanasios, and Vollmer, Waldemar

Published
2020

12. Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi‐enzyme complexes in Escherichia coli

Author

Banzhaf, Manuel, primary, Yau, Hamish CL, additional, Verheul, Jolanda, additional, Lodge, Adam, additional, Kritikos, George, additional, Mateus, André, additional, Cordier, Baptiste, additional, Hov, Ann Kristin, additional, Stein, Frank, additional, Wartel, Morgane, additional, Pazos, Manuel, additional, Solovyova, Alexandra S, additional, Breukink, Eefjan, additional, van Teeffelen, Sven, additional, Savitski, Mikhail M, additional, den Blaauwen, Tanneke, additional, Typas, Athanasios, additional, and Vollmer, Waldemar, additional

Published
2020
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13. The Two-State Prehensile Tail of the Antibacterial Toxin Colicin N

Author

Johnson, Christopher L., Solovyova, Alexandra S., Hecht, Olli, Macdonald, Colin, Waller, Helen, Grossmann, J. Günter, Moore, Geoffrey R., and Lakey, Jeremy H.

Subjects
Models, Molecular, Saccharomyces cerevisiae Proteins, Protein Conformation, Escherichia coli Proteins, Proteins, Colicins, Membrane Proteins, Porins, Elasticity, Anti-Bacterial Agents, Intrinsically Disordered Proteins, Solutions, Protein Domains, X-Ray Diffraction, Mutation, Scattering, Small Angle, Escherichia coli, Hydrodynamics, Amino Acid Sequence, Nuclear Magnetic Resonance, Biomolecular, Ultracentrifugation
Abstract

Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria. Like some other intrinsically disordered proteins in the solution state of the protein, ColN-T shows dual recognition, initially interacting with other domains of the same colicin N molecule and later, during cell killing, binding to two different receptors, OmpF and TolA, in the target bacterium. ColN-T is invisible in the high-resolution x-ray model and yet accounts for 90 of the toxin’s 387 amino acid residues. To reveal its solution structure that underlies such a dynamic and complex system, we carried out mutagenic, biochemical, hydrodynamic and structural studies using analytical ultracentrifugation, NMR, and small-angle x-ray scattering on full-length ColN and its fragments. The structure was accurately modeled from small-angle x-ray scattering data by treating ColN as a flexible system, namely by the ensemble optimization method, which enables a distribution of conformations to be included in the final model. The results reveal, to our knowledge, for the first time the dynamic structure of a colicin T-domain. ColN-T is in dynamic equilibrium between a compact form, showing specific self-recognition and resistance to proteolysis, and an extended form, which most likely allows for effective receptor binding.

Published
2017

14. Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics

Author

Robson, Timothy, Shah, Deepan S. H., Solovyova, Alexandra S., and Lakey, Jeremy H.

Subjects
gold nanoparticles, outer membrane proteins, LSPR, lateral flow assay, self-assembly, biosensing, single chain variable fragment, Article
Abstract

Functional protein–gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM) to create protein–AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpATM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur–gold bond in cysOmpATM creates a more densely populated AuNP surface than the poorly organized wtOmpATM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpATM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpATM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpATM–AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics.

Published
2018

16. The outer membrane lipoprotein NlpI nucleates hydrolases within peptidoglycan multi-enzyme complexes inEscherichia coli

Author

Banzhaf, Manuel, primary, Yau, Hamish C. L., additional, Verheul, Jolanda, additional, Lodge, Adam, additional, Kritikos, George, additional, Mateus, André, additional, Hov, Ann Kristin, additional, Stein, Frank, additional, Wartel, Morgane, additional, Pazos, Manuel, additional, Solovyova, Alexandra S., additional, Savitski, Mikhail M, additional, den Blaauwen, Tanneke, additional, Typas, Athanasios, additional, and Vollmer, Waldemar, additional

Published
2019
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18. A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Author

Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Published
2015

20. Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

Author

Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Egan, Alexander J F, Jean, Nicolas L., Koumoutsi, Alexandra, Bougault, Catherine M., Biboy, Jacob, Sassine, Jad, Solovyova, Alexandra S., Breukink, Eefjan, Typas, Athanasios, Vollmer, Waldemar, Simorre, Jean Pierre, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Egan, Alexander J F, Jean, Nicolas L., Koumoutsi, Alexandra, Bougault, Catherine M., Biboy, Jacob, Sassine, Jad, Solovyova, Alexandra S., Breukink, Eefjan, Typas, Athanasios, Vollmer, Waldemar, and Simorre, Jean Pierre

Published
2014

21. Structure and function of a spectrin-like regulator of bacterial cytokinesis

Author

Cleverley, Robert, Barrett, Jeffrey, Basle, Arnaud, Bui, Nhat Khai, Hewitt, Lorraine, Solovyova, Alexandra S, Xu, Zhi-Qiang, Daniel, Richard A, Dixon, Nicholas E, Harry, Elizabeth J, Oakley, Aaron J, Vollmer, Waldemar, Lewis, Richard J, Cleverley, Robert, Barrett, Jeffrey, Basle, Arnaud, Bui, Nhat Khai, Hewitt, Lorraine, Solovyova, Alexandra S, Xu, Zhi-Qiang, Daniel, Richard A, Dixon, Nicholas E, Harry, Elizabeth J, Oakley, Aaron J, Vollmer, Waldemar, and Lewis, Richard J

Abstract

Bacterial cell division is facilitated by a molecular machine-the divisome-that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

Published
2014

29. Structural properties of a viral orthologue of cellular CD200 protein: KSHV vOX2.

Author

Ali Amini, Abbas, Solovyova, Alexandra S., Sadeghian, Hamid, Blackbourn, David J., and Rezaee, S.A. Rahim

Subjects
*CD antigens, *KAPOSI'S sarcoma, *CELL membranes, *GLYCOPROTEINS, *CIRCULAR dichroism, *IMMUNOREGULATION
Abstract

Kaposi׳s sarcoma-associated herpesvirus (KSHV) vOX2 is a cell surface glycoprotein expressed during viral lytic replication to suppress host inflammatory reactions. Here we have characterised vOX2 with biochemical, biophysical and bioinformatics tools and as a result propose a 3-dimensional model for vOX2 based on structural and functional homology with the PD-L1 protein. To validate this model, vOX2 was characterised by analytical ultracentrifugation (AUC) and circular dichroism spectroscopy (CD). The results identified the potential glycosylation sites and revealed that vOX2 is predominantly a beta-folded molecule with an RGD adhesion motif exposed on the C-terminal domain. The protein exists in monomer–dimer equilibrium similar to its IgV-type folded homologues, with 30–36% glycosylation and the molecular weight of the extracellular fragment of molecule is 32.0–33.6 kDa, much less than 50 kDa. Thus, the structural similarity to PD-L1 verifies its immunomodulatory potential and the RGD motif suggests an adhesive capacity. [ABSTRACT FROM AUTHOR]

Published
2015
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31. Crystallization and preliminary X-ray analysis of the bacillaene synthase trans-acting acyltransferase PksC.

Author

Cuskin, Fiona, Solovyova, Alexandra S., Lewis, Richard J., and Race, Paul R.

Subjects
*CRYSTALLIZATION, *ACYLTRANSFERASES, *BACILLUS subtilis, *NONRIBOSOMAL peptide synthetases, *BIOSYNTHESIS
Abstract

The antibiotic bacillaene is biosynthesized in Bacillus subtilis by a hybrid type 1 modular polyketide synthase/nonribosomal peptide synthetase of the trans-acyltransferase ( trans-AT) class. Within this system, the essential acyl-group loading activity is provided by the action of three free-standing trans-acting acyltransferases. Here, the recombinant expression, purification and crystallization of the bacillaene synthase trans-acting acyltransferase PksC are reported. A diffraction data set has been collected from a single PksC crystal to 1.44 Å resolution and the crystal was found to belong to the orthorhombic space group P212121. [ABSTRACT FROM AUTHOR]

Published
2011
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32. Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli

Author

Banzhaf, Manuel, Yau, Hamish Cl, Verheul, Jolanda, Lodge, Adam, Kritikos, George, Mateus, André, Cordier, Baptiste, Hov, Ann Kristin, Stein, Frank, Wartel, Morgane, Pazos, Manuel, Solovyova, Alexandra S, Breukink, Eefjan, van Teeffelen, Sven, Savitski, Mikhail M, den Blaauwen, Tanneke, Typas, Athanasios, Vollmer, Waldemar, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Bacterial Cell Biology & Physiology (SILS, FNWI), European Molecular Biology Laboratory [Heidelberg] (EMBL), Newcastle University [Newcastle], University of Amsterdam [Amsterdam] (UvA), Morphogénèse et Croissance microbiennes / Microbial Morphogenesis and Growth, Institut Pasteur [Paris], Utrecht University [Utrecht], Wellcome Trust (WT). Grant Number: 101824/Z/13/ZUK Research and Innovation|Medical Research Council (MRC). Grant Number: MR/N002679/1Royal Society. Grant Number: RGS\R1\191041European Molecular Biology Laboratory (EMBL). Grant Number: 664726EC|H2020|H2020 Priority Excellent Science|H2020 European Research Council (ERC). Grant Number: 679980Agence Nationale de la Recherche (ANR). Grant Number: ANR‐10‐LABX‐62‐IBEIDVolkswagen Foundation (VolkswagenStiftung), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 679980,H2020,ERC-2015-STG,RCSB(2016), and Institut Pasteur [Paris] (IP)

Subjects
Penicillin binding proteins, binding, [SDV]Life Sciences [q-bio], peptidoglycan, medicine.disease_cause, outer membrane lipopro- tein, chemistry.chemical_compound, 0302 clinical medicine, Cell Wall, wall, 0303 health sciences, ATP synthase, biology, Escherichia coli Proteins, General Neuroscience, Signal transducing adaptor protein, Articles, N-Acetylmuramoyl-L-alanine Amidase, Microbiology, Virology & Host Pathogen Interaction, Endopeptidase, peptidoglycan Subject Category Microbiology, Biochemistry, Bacterial outer membrane, bacterial cell envelope, Lipoproteins, Article, General Biochemistry, Genetics and Molecular Biology, activator, Amidase, 03 medical and health sciences, daughter cell-separation, Multienzyme Complexes, tetratricopeptide repeat, Endopeptidases, Escherichia coli, medicine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, penicillin-binding protein, endopeptidase, outer membrane lipoprotein, 030304 developmental biology, General Immunology and Microbiology, penicillin‐binding protein, crystal-structure, Virology & Host Pathogen Interaction, chemistry, lytm-domain, murein, biology.protein, identification, Peptidoglycan, protein, 030217 neurology & neurosurgery
Abstract

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh‐like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi‐enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi‐enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases., An adaptor protein for peptidoglycan hydrolases and synthases coordinates bacterial cell wall growth.

33. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L, Bakhtina, Marina M, Becker, Donald F, Bedwell, Gregory J, Bekdemir, Ahmet, Besong, Tabot MD, Birck, Catherine, Brautigam, Chad A, Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B, Chaton, Catherine T, Cölfen, Helmut, Connaghan, Keith D, Crowley, Kimberly A, Curth, Ute, Daviter, Tina, Dean, William L, Díez, Ana I, Ebel, Christine, Eckert, Debra M, Eisele, Leslie E, Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A, Fairman, Robert, Finn, Ron M, Fischle, Wolfgang, De La Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E, Cifre, José G Hernández, Herr, Andrew B, Howell, Elizabeth E, Isaac, Richard S, Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A, Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A, Kwon, Hyewon, Larson, Adam, Laue, Thomas M, Le Roy, Aline, Leech, Andrew P, Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R, Ma, Jia, May, Carrie A, Maynard, Ernest L, Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J, Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K, Park, Jin-Ku, Pawelek, Peter D, Perdue, Erby E, Perkins, Stephen J, Perugini, Matthew A, Peterson, Craig L, Peverelli, Martin G, Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E, Raynal, Bertrand DE, Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E, Rosenberg, Rose, Rowe, Arthur J, Rufer, Arne C, Scott, David J, Seravalli, Javier G, Solovyova, Alexandra S, Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M, Streicher, Werner W, Sumida, John P, Swygert, Sarah G, Szczepanowski, Roman H, Tessmer, Ingrid, Toth, Ronald T, Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan FW, Unzai, Satoru, Gruber, Anna Vitlin, Von Hippel, Peter H, Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E, Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M, and Schuck, Peter

Subjects
Calibration, Reproducibility of Results, Ultracentrifugation, 3. Good health
Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

34. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., Schuck, Peter, Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Full Text
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35. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., Schuck, Peter, Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Full Text
View/download PDF

36. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., Schuck, Peter, Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Full Text
View/download PDF

37. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., Schuck, Peter, Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Full Text
View/download PDF

38. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author

Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., Schuck, Peter, Langowski, Jörg, Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Cölfen, Helmut, Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Díez, Ana I., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, de la Torre, José García, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Cifre, José G. Hernández, Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Mücke, Norbert, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

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39. How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity

Author

Fiona Cuskin, a, b, 1 James E. Flint, 1 Tracey M. Gloster, c, 1, 2 Carl Morland, a Arnaud Baslé, a Bernard Henrissat, d Pedro M. Coutinho, d Andrea Strazzulli, e Alexandra S. Solovyova, a Gideon J. Davies, Harry J. Gilberta, 3, Cuskin, Fiona, Flint, James E, Gloster, Tracey M, Morland, Carl, Baslé, Arnaud, Henrissat, Bernard, Coutinho, Pedro M, Strazzulli, Andrea, Solovyova, Alexandra S, Davies, Gideon J, and Gilbert, Harry J.

Subjects
Glycoside Hydrolase, Oligosaccharides, Plasma protein binding, Bacillus subtilis, Crystallography, X-Ray, Ligands, Catalysi, Oligosaccharide, Lectins, Bacteroides, Glycoside hydrolase, Polysaccharide, chemistry.chemical_classification, Multidisciplinary, biology, Chemistry, Biological Sciences, isothermal titration calorimetry, Enzymes, Biochemistry, Hydrophobic and Hydrophilic Interactions, Protein Binding, Carbohydrate, Glycoside Hydrolases, Stereochemistry, Carbohydrates, Ligand, Calorimetry, Catalysis, Fructan, Hydrophobic and Hydrophilic Interaction, Biofuel, Polysaccharides, Hydrolase, Bacillus subtili, X-ray crystallography, Kinetic, Substrate (chemistry), biology.organism_classification, Fructans, Protein Structure, Tertiary, Kinetics, Enzyme, Bacteroide, Models, Chemical, Biofuels, prebiotics, Lectin, Function (biology)
Abstract

Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two- to fivefold elevation in the activity of endo-acting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 ( Bs CBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of Bs CBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of Bs CBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1– and β-2,6–linked fructans and their respective fructooligosaccharides. Conversely, appending Bs CBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron , confers exolevanase activity on the enzyme. We propose that Bs CBM66 confers specificity for levan, a branched fructan, through an “avidity” mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.

Published
2012

40. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Author

Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, Birck C, Brautigam CA, Brennerman W, Byron O, Bzowska A, Chaires JB, Chaton CT, Cölfen H, Connaghan KD, Crowley KA, Curth U, Daviter T, Dean WL, Díez AI, Ebel C, Eckert DM, Eisele LE, Eisenstein E, England P, Escalante C, Fagan JA, Fairman R, Finn RM, Fischle W, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, Cifre JG, Herr AB, Howell EE, Isaac RS, Jao SC, Jose D, Kim SJ, Kokona B, Kornblatt JA, Kosek D, Krayukhina E, Krzizike D, Kusznir EA, Kwon H, Larson A, Laue TM, Le Roy A, Leech AP, Lilie H, Luger K, Luque-Ortega JR, Ma J, May CA, Maynard EL, Modrak-Wojcik A, Mok YF, Mücke N, Nagel-Steger L, Narlikar GJ, Noda M, Nourse A, Obsil T, Park CK, Park JK, Pawelek PD, Perdue EE, Perkins SJ, Perugini MA, Peterson CL, Peverelli MG, Piszczek G, Prag G, Prevelige PE, Raynal BD, Rezabkova L, Richter K, Ringel AE, Rosenberg R, Rowe AJ, Rufer AC, Scott DJ, Seravalli JG, Solovyova AS, Song R, Staunton D, Stoddard C, Stott K, Strauss HM, Streicher WW, Sumida JP, Swygert SG, Szczepanowski RH, Tessmer I, Toth RT 4th, Tripathy A, Uchiyama S, Uebel SF, Unzai S, Gruber AV, von Hippel PH, Wandrey C, Wang SH, Weitzel SE, Wielgus-Kutrowska B, Wolberger C, Wolff M, Wright E, Wu YS, Wubben JM, and Schuck P

Subjects
Calibration, Reproducibility of Results, Ultracentrifugation methods, Ultracentrifugation standards
Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Published
2015
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