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Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.ORCID-id: 0000-0001-7004-8251
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
Departments of Medical Genetics, Biochemistry and Chemistry, The University of Toronto, Canada.
Vise andre og tillknytning
2015 (engelsk)Inngår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, nr 3, s. 341-351Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

sted, utgiver, år, opplag, sider
Springer Netherlands, 2015. Vol. 62, nr 3, s. 341-351
Emneord [en]
CPMG, 13Cα labeling, [2-13C]-Glycerol, Excited states
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-117073DOI: 10.1007/s10858-015-9948-1ISI: 000357489200010OAI: oai:DiVA.org:liu-117073DiVA, id: diva2:805357
Merknad

At the time for thesis presentation publication was in status: Manuscript

At the time for thesis presentation name of publication was: Fractional enrichment using [2-13C]-glycerol as the carbon source facilitates measurements of excited state 13Cα chemical shifts with improved sensitivity

Tilgjengelig fra: 2015-04-15 Laget: 2015-04-15 Sist oppdatert: 2017-12-04bibliografisk kontrollert
Inngår i avhandling
1. Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
Åpne denne publikasjonen i ny fane eller vindu >>Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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. 

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press, 2015. s. 58
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1649
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-117076 (URN)10.3384/diss.diva-117076 (DOI)978-91-7519-103-4 (ISBN)
Disputas
2015-05-22, Planck, Fysikhuset, Campus Valla, Linköping, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2015-04-15 Laget: 2015-04-15 Sist oppdatert: 2019-11-15bibliografisk kontrollert

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