Intrinsically Disordered Proteins: Methods and Protocols

Part I: Sequence Properties

1. Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins

Gary W. Daughdrill

 

2. Computational Prediction of Intrinsic Disorder in Protein Sequences with the disCoP Meta-predictor

Christopher J. Oldfield, Xiao Fan, Chen Wang, A. Keith Dunker^, and Lukasz Kurgan

 

3. Computational Prediction of Disordered Protein Motifs using SLiMSuite

Richard J Edwards, Kirsti Paulsen, Carla M Aguilar Gomez, and Åsa Pérez-Bercoff

 

4. How to Annotate and Submit a Short Linear Motif to the Eukaryotic Linear Motif Resource

Marc Gouw, Jesús AlvaradoValverde, Jelena Calyseva, Francesca Diella, Manjeet Kumar, Sushama Michael, Kim Van Roey, Holger Dinkel, and Toby J. Gibson

 

5. Analyzing the Sequences of Intrinsically Disordered Regions with CIDER and localCIDER

 Garrett M. Ginell and Alex S. Holehouse

 

6. Exploring Protein Intrinsic Disorder with MobiDB

Alexander Miguel Monzon, András Hatos, Marco Necci, Damiano Piovesan, and Silvio C. E. Tosatto

 

Part II: Evolution

7. An Easy Protocol for Evolutionary Analysis of Intrinsically Disordered Proteins
Janelle Nunez-Castilla and Jessica Siltberg-Liberles

 

Part III: Production

8. Expression and Purification of an Intrinsically Disordered Protein

Karamjeet K. Singh and Steffen P. Graether

 

9. Production of Intrinsically Disordered Proteins for Biophysical Studies; Tips and Tricks

Christian Parsbæk Pedersen, Pernille Seiffert, Inna Brakti, and Katrine Bugge

 

10. Recombinant Production of Monomeric Isotope-Enriched Aggregation-Prone Peptides: Polyglutamine Tracts and Beyond

Albert Escobedo, Giulio Chiesa, and Xavier Salvatella

 

11. Cell-free Protein Synthesis of Small Intrinsically Disordered Proteins for NMR Spectroscopy

Linnéa Isaksson and Anders Pedersen

 

Part IV: Dynamics, Ensembles, and Structures

12. Structural Analyses of Intrinsically Disordered Proteins by Small-Angle X-ray Scattering

Amin Sagar, Dmitri Svergun, and Pau Bernadó

 

13. Determining Rg of IDPs from SAXS data
Ellen Rieloff and Marie Skepö

 

14. Obtaining Hydrodynamic Radii of Intrinsically Disordered Protein Ensembles by Pulsed Field Gradient NMR Measurements

Sarah Leeb and Jens Danielsson

 

15. Quantitative Protein Disorder Assessment using NMR Chemical Shifts

Jakob T. Nielsen and Frans A. A. Mulder

 

16. Determination of pK_a Values in Intrinsically Disordered Proteins

Brandon Payliss and Anthony Mittermaier

 

17. Paris-DÉCOR: A Protocol for the Determination of Fast Protein Backbone Amide Hydrogen Exchange Rates

Rupashree Dass and Frans A.A. Mulder

 

Part V: Ensembles by Computation

18. Predicting Conformational Properties of Intrinsically Disordered Proteins from Sequence

Kiersten M. Ruff

 

19. Enhanced Molecular Dynamics Simulations of Intrinsically Disordered Proteins

Matteo Masetti, Mattia Bernetti, and Andrea Cavalli

 

20. Computational Protocol for Determining Conformational Ensembles of Intrinsically Disordered Proteins

Robert B. Best

 

21. Computing, Analyzing and Comparing the Radius of Gyration and Hydrodynamic Radius in Conformational Ensembles of Intrinsically Disordered Proteins

Mustapha Carab Ahmed, Ramon Crehuet, and Kresten Lindorff-Larsen

 

Part VI: Determinants of Interactions

22. Binding Thermodynamics to Intrinsically Disordered Protein Domains

Arne Schön and Ernesto Freire

 

23. Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements

Samuel Sparks, Ryo Hayama, Michael P. Rout, and David Cowburn

 

24. NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

Christopher A. Waudby and John Christodoulou

 

25. Measuring Effective Concentrations Enforced by Intrinsically Disordered Linkers

Charlotte S. Sørensen and Magnus Kjaergaard

 

26. Determining the Protective Activity of IDPs under Partial Dehydration and Freeze-thaw Conditions 

David F. Rendón-Luna, Paulette S. Romero-Pérez, Cesar L. Cuevas-Velazquez, José L. Reyes, and Alejandra A. Covarrubias

 

27. Screening Intrinsically Disordered Regions for Short Linear Binding Motifs

Muhammad Ali, Leandro Simonetti, and Ylva Ivarsson

 

Part VII: Interactions on Surfaces

28. Probing IDP Interactions with Membranes by Fluorescence Spectroscopy

Diana Acosta, Tapojyoti Das, and David Eliezer

 

29. Protocol for Investigating the Interactions between Intrinsically Disordered Proteins and Membranes by Neutron Reflectometry

Alessandra Luchini and Lise Arleth

 

30. Interactions of IDPs with Membranes Using Dark State Exchange NMR Spectroscopy

Tapojyoti Das, Diana Acosta, and David Eliezer

 

Part IIX: Binding Kinetics and Mechanisms 31. Determination of Binding Kinetics of Intrinsically Disordered Proteins by Surface Plasmon Resonance Julie M. Leth and Michael Ploug

 

32. Measuring and Analysing Binding Kinetics of Coupled Folding and Binding Reactions under Pseudo-first Order Conditions

Kristine Steen Jensen

 

33. Understanding Binding Induced Folding by Temperature Jump

Angelo Toto, Francesca Troilo, Francesca Malagrinò, and Stefano Gianni

 

34. Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR spectroscopy

Ke Yang, Munehito Arai, and Peter E. Wright

 

Part IX: Higher Order-Phase Separation and Fibrillation

35. Determination of Protein Phase Diagrams by Centrifugation

Nicole M. Milkovic and Tanja Mittag

 

36. In vitro Transition Temperature Measurement of Phase Separating Proteins by Microscopy

Jack Holland, Michael D. Crabtree, and Timothy J. Nott

 

37. Walking along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering

Ivan Peran, Erik W. Martin, and Tanja Mittag

 

38. Expression and Purification of Intrinsically Disordered Abeta Peptide and Setup of Reproducible Aggregation Kinetics Experiment

Sara Linse

 

39. Measuring Interactions between Tau and Aggregation Inducers with Single Molecule Förster Resonance Energy Transfer

Sanjula P. Wickramasinghe and Elizabeth Rhoades

 

Part X: Modification and Targeting

40. Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins using Phos-tag SDS-PAGE

Mihkel Örd and Mart Loog

 

41. Multiple Site-specific Phosphorylation of IDPs Monitored by NMR

Manon Julien, Chafiaa Bouguechtouli, Ania Alik, Rania Ghouil, Sophie Zinn-Justin, and François-Xavier Theillet

 

42. Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins using Quantitative Mass-Spectrometry

Ervin Valk, Artemi Maljavin, and Mart Loog

 

43. Targeting an Intrinsically Disordered Protein by Covalent Modification

Hung Nguyen, Péter Ábrányi-Balogh, László Petri, Attila Mészáros, Kris Pauwels, Guy Vandenbussche, György Miklós Keser , and Peter Tompa

 

Part XI: In Cell and Interactomes

44. Recording in-cell NMR-spectra in Living Mammalian Cells

Irena Matecko-Burmann and Björn M. Burmann

 

45. In-cell NMR of Intrinsically Disordered Proteins in Mammalian Cells

Juan A. Gerez, Natalia C. Prymaczok, and Roland Riek

 

46. Analyzing IDPs in Interactomes

Vladimir N. Uversky