| 1. |
- Auer, Renate, et al.
(författare)
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Measuring the Signs of H-1(alpha) Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R-1 rho NMR Spectroscopy
- 2009
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Ingår i: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:31, s. 10832-10833
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical, shift differences between NMR probes in the exchanging states, vertical bar Delta(pi)vertical bar, are also extracted. Herein, we present a simple experiment for obtaining the sign of H-1(alpha) Delta(pi) values by measuring off-resonance H-1(alpha) decay rates, R-1 rho, using weak proton spin-lock fields. A pair of R-1 rho values is measured with a spin-lock field applied vertical bar Delta omega vertical bar downfield and upfield of the major-state peak. In many cases, these two relaxation rates differ substantially, with the larger one corresponding to the case where the spin-lock field coincides with the resonance frequency of the probe in the minor state. The utility of the methodology is demonstrated first on a system involving protein ligand exchange and subsequently on an SH3 domain exchanging between a folded state and its on-pathway folding intermediate. With this experiment, it thus becomes possible to determine H-1(alpha) chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.
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| 2. |
- Hansen, D. Flemming, et al.
(författare)
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Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: How well can we do?
- 2008
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society. - 0002-7863 .- 1520-5126. ; 130:8, s. 2667-2675
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Tidskriftsartikel (refereegranskat)abstract
- Carr−Purcell−Meiboom−Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone 13Cα and 13CO relaxation dispersion profiles in proteins without complications from 13C−13C couplings. Such measurements are presented here, along with those that probe exchange using 15N and 1HN nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.
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| 3. |
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| 4. |
- Neudecker, Philipp, et al.
(författare)
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Relaxation Dispersion NMR Spectroscopy as a Tool for Detailed Studies of Protein Folding
- 2009
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 96:6, s. 2045-2054
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Tidskriftsartikel (refereegranskat)abstract
- Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a "high-resolution" picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.
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| 5. |
- Neudecker, Philipp, et al.
(författare)
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Structure of an Intermediate State in Protein Folding and Aggregation
- 2012
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 336:5079, s. 362-366
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Tidskriftsartikel (refereegranskat)abstract
- Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal beta strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.
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