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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 http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. 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
Identifiers
URN: urn:nbn:se:liu:diva-115010DOI: 10.1371/journal.pcbi.1004022ISI: 000349309400013PubMedID: 25569628OAI: oai:DiVA.org:liu-115010DiVA: diva2:793678
Note

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

Available from: 2015-03-09 Created: 2015-03-06 Last updated: 2017-12-04
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.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1649
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-117076 (URN)10.3384/diss.diva-117076 (DOI)978-91-7519-103-4 (ISBN)
Public defence
2015-05-22, Planck, Fysikhuset, Campus Valla, Linköping, 09:15 (English)
Opponent
Supervisors
Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-23Bibliographically approved
2. Coding to cure: NMR and thermodynamic software applied to congenital heart disease research
Open this publication in new window or tab >>Coding to cure: NMR and thermodynamic software applied to congenital heart disease research
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regardless of scientific field computers have become pivotal tools for data analysis and the field of structural biology is not an exception. Here, computers are the main tools used for tasks including structural calculations of proteins, spectral analysis of nuclear magnetic resonance (NMR) spectroscopy data and fitting mathematical models to data. As results reported in papers heavily rely on software and scripts it is of key importance that the employed computational methods are robust and yield reliable results. However, as many scientific fields are niched and possess a small potential user base the task to develop necessary software often falls on researchers themselves. This can cause divergence when comparing data analyzed by different measures or by using subpar methods. Therein lies the importance of development of accurate computational methods that can be employed by the scientific community.

The main theme of this thesis is software development applied to structural biology, with the purpose to aid research in this scientific field by speeding up the process of data analysis as well as to ensure that acquired data is properly analyzed. Among the original results of this thesis are three user-friendly software:

COMPASS - a resonance assignment software for NMR spectroscopy data capable of analyzing chemical shifts and providing the user with suggestions to potential resonance assignments, based on a meticulous database comparison.

CDpal - a curve fitting software used to fit thermal and chemical denaturation data of proteins acquired by circular dichroism (CD) spectroscopy or fluorescence spectroscopy.

PINT - a line shape fitting and downstream analysis software forNMRspectroscopy data, designed with the important purpose to easily and accurately fit peaks in NMR spectra and extract parameters such as relaxation rates, intensities and volumes of peaks.

This thesis also describes a study performed on variants of the life essential regulatory protein calmodulin that have been associated with the congenital life threatening heart disease long QT syndrome (LQTS). The study provided novel insights revealing that all variants are distinct from the wild type in regards to structure and dynamics on a detailed level; the presented results are useful for the interpretation of results from protein interaction studies. The underlying research of this paper makes use of all three developed software, which validates that all developed methods fulfil a scientific purpose and are capable of producing solid results.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2017. 76 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1882
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:liu:diva-142785 (URN)10.3384/diss.diva-142785 (DOI)9789176854495 (ISBN)
Public defence
2017-11-24, Planck, Fysikhuset, Campus Valla, Linköping, 10:00 (English)
Opponent
Supervisors
Available from: 2017-11-03 Created: 2017-11-03 Last updated: 2017-12-01Bibliographically approved

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Niklasson, MarkusAhlner, AlexandraAndrésen, CeciliaLundström, Patrik

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