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Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
University of Edinburgh, Scotland.
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2015 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 11, no 1, e1004022- p.Article in journal (Refereed) Published
Abstract [en]

The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

Place, publisher, year, edition, pages
Public Library of Science , 2015. Vol. 11, no 1, e1004022- p.
National Category
Chemical Sciences
URN: urn:nbn:se:liu:diva-115010DOI: 10.1371/journal.pcbi.1004022ISI: 000349309400013PubMedID: 25569628OAI: diva2:793678

Funding Agencies|Swedish Research Council [Dnr. 2012-5136]

Available from: 2015-03-09 Created: 2015-03-06 Last updated: 2015-04-15
In thesis
1. Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
Open this publication in new window or tab >>Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.

To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.

To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism. 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 58 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1649
National Category
Chemical Sciences
urn:nbn:se:liu:diva-117076 (URN)10.3384/diss.diva-117076 (DOI)978-91-7519-103-4 (print) (ISBN)
Public defence
2015-05-22, Planck, Fysikhuset, Campus Valla, Linköping, 09:15 (English)
Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-23Bibliographically approved

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