| 1. |
- Auer, Renate, et al.
(författare)
-
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
-
Ingår i: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:31, s. 10832-10833
-
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.
|
|
| 2. |
- Eisenmesser, Elan Z, et al.
(författare)
-
Intrinsic dynamics of an enzyme underlies catalysis
- 2005
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 438, s. 117-21
-
Tidskriftsartikel (refereegranskat)abstract
- A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis1, 2, 3, 4, 5. The concept of conformational substates has been described6; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis–trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously3, 7, 8, 9, 10, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
|
|
| 3. |
- Hansen, D. Flemming, et al.
(författare)
-
Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: How well can we do?
- 2008
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society. - 0002-7863 .- 1520-5126. ; 130:8, s. 2667-2675
-
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.
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
- Korzhnev, Dmitry M., et al.
(författare)
-
The folding pathway of an FF domain : Characterization of an on-pathway intermediate state under folding conditions by N-15, C-13(alpha) and C-13-methyl relaxation dispersion and H-1/(2) H-exchange NMR Spectroscopy
- 2007
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 372:2, s. 497-512
-
Tidskriftsartikel (refereegranskat)abstract
- The FF domain from the human protein HYPA/FBP11 folds via a lowenergy on-pathway intermediate (. Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by N-15, C-13(alpha) and methyl C-13 Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by 15N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U-I-F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone N-15, C-13(alpha) and side-chain methyl 13C chemical shifts, with further information from protection factors for the backbone amide groups from H-1/(2) H-exchange. Notably, helices H1-H3 are at least partially formed in 1, while helix H4 is largely disordered. Chemical shift differences for the methyl 13 C nuclei suggest a paucity of stable, native-like hydrophobic interactions in 1. These data are consistent with (D-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and (1) data can elucidate whole experimental folding pathways.
|
|
| 7. |
- Lundström, Patrik, 1971-, et al.
(författare)
-
A single-quantum methyl C-13-relaxation dispersion experiment with improved sensitivity
- 2007
-
Ingår i: Journal of Biomolecular NMR. - : Springer. - 0925-2738 .- 1573-5001. ; 38:1, s. 79-88
-
Tidskriftsartikel (refereegranskat)abstract
- A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.
|
|
| 8. |
- Lundström, Patrik, 1971-, et al.
(författare)
-
Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy
- 2009
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:5, s. 1915-1926
-
Tidskriftsartikel (refereegranskat)abstract
- Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy can provide detailed information about low populated, invisible states of protein molecules, including backbone chemical shifts of the invisible conformer and bond vector orientations that can be used as structural constraints. Notably, the measurement of H-1(alpha) chemical shifts in excited protein states has not been possible to date because, in the absence of suitable labeling, the homonuclear proton scalar coupling network in side chains of proteins leads to a significant degradation in the performance of proton-based relaxation dispersion experiments. Here we have overcome this problem through a labeling scheme in which proteins are prepared with U-H-2 glucose and 50% D2O/50% H2O that results in cleuteration levels of between 50-88% at the C-beta carbon. Effects from residual H-1(alpha)-H-1(beta) scalar couplings can be suppressed through a new NMR experiment that is presented here. The utility of the methodology is demonstrated on a ligand binding exchanging system and it is shown that H-1(alpha) chemical shifts extracted from dispersion profiles are, on average, accurate to 0.03 ppm, an order of magnitude better than they can be predicted from structure using a database approach. The ability to measure H-1(alpha) chemical shifts of invisible conformers is particularly important because such shifts are sensitive to both secondary and tertiary structure. Thus, the methodology presented is a valuable addition to a growing list of experiments for characterizing excited protein states that are difficult to study using the traditional techniques of structural biology.
|
|
| 9. |
- Lundström, Patrik, et al.
(författare)
-
Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Ca and side-chain methyl positions in protein
- 2007
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 38:3, s. 199-212
-
Tidskriftsartikel (refereegranskat)abstract
- A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.
|
|
| 10. |
- Lundström, Patrik, 1971-, et al.
(författare)
-
Fractional C-13 enrichment of isolated carbons using [1-C-13]- or [2-C-13]-glucose facilitates the accurate measurement of dynamics at backbone C-alpha and side-chain methyl positions in proteins
- 2007
-
Ingår i: Journal of Biomolecular NMR. - : Springer. - 0925-2738 .- 1573-5001. ; 38:3, s. 199-212
-
Tidskriftsartikel (refereegranskat)abstract
- A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.
|
|
| 11. |
- Lundström, Patrik, et al.
(författare)
-
Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy.
- 2009
-
Ingår i: Nature protocols. - : Springer Science and Business Media LLC. - 1750-2799 .- 1754-2189. ; 4:11, s. 1641-1648
-
Tidskriftsartikel (refereegranskat)abstract
- The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state.
|
|
| 12. |
- Lundström, Patrik, 1971-, et al.
(författare)
-
Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively C-13 labeled samples
- 2008
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 42:1, s. 35-47
-
Tidskriftsartikel (refereegranskat)abstract
- Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated C-13 spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (CO)-C-13 experiments however the large separation between (CO)-C-13 and C-13(alpha) chemical shifts offers hope that robust (CO)-C-13 dispersion profiles can be recorded on uniformly C-13 labeled samples, leading to the extraction of accurate (CO)-C-13 chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-C-13]-pyruvate and (NaHCO3,)-C-13 or uniformly labeled, generated from C-13-glucose. Very similar (CO)-C-13 chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.
|
|
| 13. |
- Lundström, Patrik, et al.
(författare)
-
Measuring C-13(beta) chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy
- 2009
-
Ingår i: JOURNAL OF BIOMOLECULAR NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 44:3, s. 139-155
-
Tidskriftsartikel (refereegranskat)abstract
- A labeling scheme is introduced that facilitates the measurement of accurate C-13(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of C-13 enrichment (30-40%) at C-beta side-chain carbon positions for 15 of the amino acids with little C-13 label at positions one bond removed (a parts per thousand 5%). A pair of samples are produced using [1-C-13]-glucose/(NaHCO3)-C-12 or [2-C-13]-glucose as carbon sources with isolated and enriched (andgt; 30%) C-13(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of C-13(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.
|
|
| 14. |
- Neudecker, Philipp, et al.
(författare)
-
Relaxation Dispersion NMR Spectroscopy as a Tool for Detailed Studies of Protein Folding
- 2009
-
Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 96:6, s. 2045-2054
-
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.
|
|
| 15. |
|
|
| 16. |
- Vallurupalli, Pramodh, et al.
(författare)
-
CPMG relaxation dispersion NMR experiments measuring glycine H-1(alpha) and C-13(alpha) chemical shifts in the 'invisible' excited states of proteins
- 2009
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 45:1-2, s. 45-55
-
Tidskriftsartikel (refereegranskat)abstract
- Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (> 0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of H-1(alpha) and C-13(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the H-1(alpha), C-13(alpha) spins form a three-spin system that is more complex than the AX H-1(alpha)-C-13(alpha) spin systems in the other amino acids. Here pulse sequences for recording H-1(alpha) and C-13(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which H-1(alpha), C-13(alpha) chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.
|
|
| 17. |
- Zhuravleva, Anastasia, 1979, et al.
(författare)
-
Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study.
- 2007
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836. ; 367:4, s. 1079-92
-
Tidskriftsartikel (refereegranskat)abstract
- NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.
|
|
