CSSB Centre for Structural Systems Biology
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Protein Characterisation

  • Quality control for structural studies
  • MS intact mass
  • Protein-protein interaction
  • Time-resolved experiments

About

Available Instruments

  • VP ITC, MicroCal - Isothermal Titration Calorimetry, study of biomolecular interactions in solution
  • PEAQ-ITC, MicroCal - Isothermal Titration Calorimetry, study of biomolecular interactions in solution
  • Monolith NT.115 (Microscale thermophoresis), NanoTemper - measuring binding affinities (labelling of 1 partner required)
  • Monolith NT. Label-free (Microscale thermophoresis), NanoTemper - measuring binding affinities (label-free)
  • DLS Nanostar (Dynamic light scattering), Malvern - particle size measurements. Addressing dispersity/ aggregational state of samples.
  • Biacore T200, GE - SPR, interaction of biomolecules and kinetics
  • CD (Circular Dichroism), Applied Photophysics - Secondary structure analysis.
  • Prometheus NT.48 with backscattering optics (nano-Differential Scanning Fluorimetry technology), NanoTemper - study protein stability and protein aggregation.
  • FTIR (Fourier Transform Infrared Spectrometer) Bruker - Protein spectrum. Analysing protein signals from formulation components.
  • MALDI TOF, Bruker/CovalX - Determination of the molecular mass of protein samples and peptides resulting from proteolytic digestions. CovalX HM4 High-Mass System enables protein complex analysis and intact protein analysis.
  • Octet RED96 System, PAL ForteBio - 96-well characterization of protein-protein and protein-small molecule binding kinetics, determination of protein concentrations.
  • SPARK 20M Plate reader, TECAN - Detection Absorbance, Fluorescence intensity, FP, TRF, FRET, TR-FRET, luminescence (flash, glow, multi-colour), Alpha Technology, Automated live cell imaging
  • Stopped-Flow, Applied Photophysics - Time resolved experiments
  • Mass photometer, Refeyn - can monitor protein-protein interactions at a single-molecule level with high-sensitivity and can at the same time determine molecular weight of proteins and protein complexes with a high dynamic range and great accuracy. The mass photometer is an ideal tool for quality control in the protein structural analysis workflow as it can assess the molecular mass and the oligomerisation status of a sample in one measurement.
  • SSM-based electrophysiology, SURFE²R N1 - designed for the measurements of electrogenic transporters (symporters, exchangers and uniporters) and pumps. Usually these proteins have low turnover rates compared to ion channels. SURFE²R technology compensates for that with a large sensor size which allows for the measurement of up to 109 transporters at the same time to yield the best signal to noise ratio.

 

Services

We support users with the design, execution and data analysis of biophysical experiments aimed at the characterization of proteins, protein complexes and interactions between proteins and other types of molecules.

The PC facility offers a variety of services related to protein characterization:

  • Quality control for structural studies
  • MS intact mass
  • Protein-protein interaction

 

Further Information

Our protein characterization platform located at the CSSB includes several biophysical instruments available for in-house and external users. We offer services for protein quality control in addition to characterization techniques for studying the interaction of biomolecules. Techniques available: MS MALDI coupled to a high mass detector, Thermofluor (TF), Circular Dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Isothermal Titration Calorimetry (ITC), nano Differential Scanning Fluorimetry (nDSF), Surface Plasmon Resonance (SPR), Biolayer interferometry (Octet), Microscale Thermophoresis (MST) and Dynamic Light Scattering (DLS).

Access & Team

Access

The PC facility is a fee-for-service (non-profit) technology platform operated by EMBL in Hamburg. No service fees are charged for usage of this equipment by EMBL, CSSB and beamline users. An online system (iLAB) to streamline the process of booking instruments, ordering and billing core service requests is in place and in routine use.

All users must follow a safety training before working in our laboratories. Complete the SPC safety briefing and examination before your visit though SMIS. Please go to the Safety tab and select "Standard Training"

Our acknowledgement policy

Please acknowledge any substantial contributions of the SPC Facility to your research in publications. Please state the following in the acknowledgement section of your publication: “We acknowledge technical support by the SPC facility at EMBL Hamburg”.

Please don't forget to let us know when you publish papers that acknowledge us. This will help us keep track and to justify the existence of the facility to funding bodies.

Team

3

Facility Head

Dr. Maria Garcia Alai
Phone:+49 040 8990 2145

E-Mail

Staff Scientist

Angelica Struve Garcia
Phone:+49 40 8998 87655

E-Mail

Staff Scientist

Dr. Stephan Niebling
Phone:+49 40 8998 87655

E-Mail

Address

CSSB Centre for Structural Systems Biology
c/o Deutsches Elektronen-Synchrotron DESY
Notkestraße 85, Building 15
D-22607 Hamburg

Facility Operator: European Molecular Biology Laboratory (EMBL)

Publications

2021

Lorenzo R, Defelipe LA, Aliperti L, Niebling S, Custódio TF, Löw C, Schwarz JJ, Remans K, Craig PO, Otero LH, Klinke S, García-Alai M, Sánchez IE, Alonso LG (2021) Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif. J Biol Chem.101175. doi: 10.1016/j.jbc.2021.101175.

López-Méndez B, Baron B, Brautigam CA, Jowitt TA, Knauer SH, Uebel S, Williams MA, Sedivy A, Abian O, Abreu C, Adamczyk M, Bal W, Berger S, Buell AK, Carolis C, Daviter T, Fish A, Garcia-Alai M, Guenther C, Hamacek J, Holková J, Houser J, Johnson C, Kelly S, Leech A, Mas C, Matulis D, McLaughlin SH, Montserret R, Nasreddine R, Nehmé R, Nguyen Q, Ortega-Alarcón D, Perez K, Pirc K, Piszczek G, Podobnik M, Rodrigo N, Rokov-Plavec J, Schaefer S, Sharpe T, Southall J, Staunton D, Tavares P, Vanek O, Weyand M, Wu D (2021) Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study. Eur Biophys J. 50(3-4):411-427. doi:10.1007/s00249-021-01532-6.

de Marco A, Berrow N, Lebendiker M, Garcia-Alai M, Knauer SH, Lopez-Mendez B, Matagne A, Parret A, Remans K, Uebel S, Raynal B (2021) Quality control of protein reagents for the improvement of research data reproducibility. Nat Commun.12(1):2795. doi:10.1038/s41467-021-23167-z.

Niebling S, Burastero O, Bürgi J, Günther C, Defelipe LA, Sander S, Gattkowski E, Anjanappa R, Wilmanns M, Springer S, Tidow H, García-Alai M (2021) FoldAffinity: binding affinities from nDSF experiments. Sci Rep. 11(1):9572. doi:10.1038/s41598-021-88985-z.

Berrow N, de Marco A, Lebendiker M, Garcia-Alai M, Knauer SH, Lopez-Mendez B, Matagne A, Parret A, Remans K, Uebel S, Raynal B (2021) Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey. Eur Biophys J. 50(3-4):453-460. doi:10.1007/s00249-021-01528-2.

Winkel Missel J, Salustros N, Ramos Becares E, Hyld Steffen J, Gerdt Laursen A, Struve Garcia A, Garcia-Alai MM, Kolar Č, Gourdon P, Gotfryd K (2021) Cyclohexyl-α maltoside as a highly efficient tool for membrane protein studies. Current Research in Structural Biology. https://doi.org/10.1016/j.crstbi.2021.03.002.

Lizarrondo J, Klebl DP, Niebling S, Abella M, Schroer MA, Mertens HDT, Veith K, Thuenauer R, Svergun DI, Skruzny M, Sobott F, Muench SP, Garcia-Alai MM (2021) Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis. Nat Commun.12(1):2889. doi: 10.1038/s41467-021-23151-7.

Günther S, Reinke PYA, Fernández-García Y, Lieske J, Lane TJ, Ginn HM, Koua FHM, Ehrt C, Ewert W, Oberthuer D, Yefanov O, Meier S, Lorenzen K, Krichel B, Kopicki JD, Gelisio L, Brehm W, Dunkel I, Seychell B, Gieseler H, Norton-Baker B, Escudero-Pérez B, Domaracky M, Saouane S, Tolstikova A, White TA, Hänle A, Groessler M, Fleckenstein H, Trost F, Galchenkova M, Gevorkov Y, Li C, Awel S, Peck A, Barthelmess M, Schluenzen F, Lourdu Xavier P, Werner N, Andaleeb H, Ullah N, Falke S, Srinivasan V, França BA, Schwinzer M, Brognaro H, Rogers C, Melo D, Zaitseva-Doyle JJ, Knoska J, Peña-Murillo GE, Mashhour AR, Hennicke V, Fischer P, Hakanpää J, Meyer J, Gribbon P, Ellinger B, Kuzikov M, Wolf M, Beccari AR, Bourenkov G, von Stetten D, Pompidor G, Bento I, Panneerselvam S, Karpics I, Schneider TR, Garcia-Alai MM, Niebling S, Günther C, Schmidt C, Schubert R, Han H, Boger J, Monteiro DCF, Zhang L, Sun X, Pletzer-Zelgert J, Wollenhaupt J, Feiler CG, Weiss MS, Schulz EC, Mehrabi P, Karničar K, Usenik A, Loboda J, Tidow H, Chari A, Hilgenfeld R, Uetrecht C, Cox R, Zaliani A, Beck T, Rarey M, Günther S, Turk D, Hinrichs W, Chapman HN, Pearson AR, Betzel C, Meents A. (2021) X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease. Science. 2021 Apr 2:eabf7945. doi: 10.1126/science.abf7945.

2020

Heidemann J, Kölbel K, Konijnenberg A, Van Dyck J, Garcia-Alai M, Meijers R, Sobott F, Uetrecht C. (2020) Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies. Int J Mass Spectrom. 2019 Oct 18;447:116240. doi: 10.1016/j.ijms.2019.116240.

Anjanappa R, Garcia-Alai M, Kopicki JD, Lockhauserbäumer J, Aboelmagd M, Hinrichs J, Nemtanu IM, Uetrecht C, Zacharias M, Springer S, Meijers R (2020) Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection. Nat Commun. 11;11(1):1314. doi: 10.1038/s41467-020-14862-4.

2019

Saini SK, Tamhane T, Anjanappa R, Saikia A, Ramskov S, Donia M, Svane IM, Jakobsen SN, Garcia-Alai M, Zacharias M, Meijers R, Springer S, Hadrup SR (2019) Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells. Sci Immunol 410.1126/sciimmunol.aau9039 doi: 10.1126/sciimmunol.aau9039

Moritz A, Anjanappa R, Wagner C, Bunk S, Hofmann M, Pszolla G, Saikia A, Garcia-Alai M, Meijers R, Rammensee HG, Springer S, Maurer D (2019) High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules. Sci Immunol 410.1126/sciimmunol.aav0860. doi: 10.1126/sciimmunol.aav0860.

Kotov V, Bartels K, Veith K, Josts I, Subhramanyam UKT, Gunther C, Labahn J, Marlovits TC, Moraes I, Tidow H, Low C, Garcia-Alai MM (2019) High-throughput stability screening for detergent-solubilized membrane proteins. Sci Rep 9: 10379.doi: 10.1038/s41598-019-46686-8

Gattkowski E, Johnsen A, Bauche A, Möckl F, Kulow F, Garcia Alai M, Rutherford TJ, Fliegert R, Tidow H. (2019) Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2. Biochim Biophys Acta Mol Cell Res. 2019 Jul;1866(7):1162-1170. doi: 10.1016/j.bbamcr.2018.12.010.

2018

Garcia-Alai MM, Heidemann J, Skruzny M, Gieras A, Mertens HDT, Svergun DI, Kaksonen M, Uetrecht C, Meijers R (2018) Epsin and Sla2 form assemblies through phospholipid interfaces. Nat Commun 9: 328 doi: 10.1038/s41467-017-02443-x

Molina IG, Josts I, Almeida Hernandez Y, Esperante S, Salgueiro M, Garcia Alai MM, de Prat-Gay G, Tidow H. (2018) Structure and stability of the Human respiratory syncytial virus M 2-1 RNA-binding core domain reveals a compact and cooperative folding unit. Acta Crystallogr F Struct Biol Commun. 2018 Jan 1;74(Pt 1):23-30. doi: 10.1107/S2053230X17017381.

 

Research

The PC facility is involved in the following research projects:

X-ray based screen of drugs against SARS-CoV-2
The project aims at performing high-throughput X-ray crystallographic screening experiments of several thousand compounds in complex with protein targets at the PETRA III synchrotron, to identify potential drug candidates, which will be subsequently tested in protein activity assays and finally virus cell cultures. The PC and HTX facilities will be used to further characterize drug/target complexes biophysically.
CSSB PC and HTX facilities: Maria Garcia Alai (EMBL)
Scientific Leadership: Alke Meents (DESY)