| 1. |
- Born, Alexandra, et al.
(författare)
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Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery
- 2019
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Ingår i: Biomolecular NMR Assignments. - : SPRINGER. - 1874-2718 .- 1874-270X. ; 13:1, s. 85-89
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Tidskriftsartikel (refereegranskat)abstract
- Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain H-1, C-13 and N-15 NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.
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| 2. |
- Chi, Celestine N., et al.
(författare)
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NOE-derived methyl distances from a 360 kDa proteasome complex
- 2018
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 24:9, s. 2270-2276
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear magnetic resonance spectroscopy is the prime tool to probe structure and dynamics of biomolecules at atomic resolution. A serious challenge for that method is the size limit imposed on molecules to be studied. Standard studies are typically restricted to ca. 30-40 kDa. More recent developments lead to spin relaxation measurements in methyl groups in single proteins or protein complexes as large as a mega-Dalton, which directly allow the extraction of angular information or experiments with paramagnetic samples. However, these probes are all of indirect nature in contrast to the most intuitive and easy-to-interpret structural/dynamics restraint, the internuclear distance, which can be measured by nuclear Overhauser enhancement (NOE). Here, we demonstrate time-averaged distance measurements on the 360 kDa half proteasome from Thermoplasma acidophilium. The approach is based on exact quantification of the NOE (eNOE). Our findings open up an avenue for such measurements on very large molecules. These restraints will help in a detailed determination of conformational changes upon perturbation such as ligand binding.
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| 3. |
- Chi N, Celestine, 1978-, et al.
(författare)
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A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution.
- 2015
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:40, s. 11657-61
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Tidskriftsartikel (refereegranskat)abstract
- For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60-70 % of the side-chain conformations of cyclophilin appear to be correlated.
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| 4. |
- Chi N, Celestine, 1978-, et al.
(författare)
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Extending the eNOE data set of large proteins by evaluation of NOEs with unresolved diagonals.
- 2015
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 62:1, s. 63-9
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Tidskriftsartikel (refereegranskat)abstract
- The representation of a protein's spatial sampling at atomic resolution is fundamental for understanding its function. NMR has been established as the best-suited technique toward this goal for small proteins. However, the accessible information content rapidly deteriorates with increasing protein size. We have recently demonstrated that for small proteins distance restraints with an accuracy smaller than 0.1 Å can be obtained by replacing traditional semi-quantitative Nuclear Overhauser Effects (NOEs) with exact NOEs (eNOE). The high quality of the data allowed us to calculate structural ensembles of the small model protein GB3 consisting of multiple rather than a single state. The analysis has been limited to small proteins because NOEs of spins with unresolved diagonal peaks cannot be used. Here we propose a simple approach to translate such NOEs into correct upper distance restraints, which opens access to larger biomolecules. We demonstrate that for 16 kDa cyclophilin A the collection of such restraints extends the original 1254 eNOEs to 3471.
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| 5. |
- Jemth, Per, et al.
(författare)
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Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins
- 2018
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 4:10
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Tidskriftsartikel (refereegranskat)abstract
- In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity Cambrian-like [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger Ordovician-Silurian fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.
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| 6. |
- Nichols, Parker J., et al.
(författare)
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Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA
- 2018
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Ingår i: ChemBioChem. - : WILEY-V C H VERLAG GMBH. - 1439-4227 .- 1439-7633. ; 19:16, s. 1695-1701
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Forskningsöversikt (refereegranskat)abstract
- Distance-dependent nuclear Overhauser enhancements (NOEs) are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semiquantitative upper distance bounds, and this discards the wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond timescale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multistate ensembles of proteins up to approximately 150 residues. Here, we briefly revisit eNOE methodology and present two new directions for the use of eNOEs: applications to large proteins and RNA.
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| 7. |
- Nichols, Parker J., et al.
(författare)
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The Exact Nuclear Overhauser Enhancement : Recent Advances
- 2017
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 22:7
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Forskningsöversikt (refereegranskat)abstract
- Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.
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| 8. |
- Strotz, Dean, et al.
(författare)
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eNORA2 Exact NOE Analysis Program
- 2017
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 13:9, s. 4336-4346
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Tidskriftsartikel (refereegranskat)abstract
- We have recently developed an NMR protocol to extract exact distances between nuclei in proteins from an exact interpretation of NOESY buildup intensities (eNOEs). This enabled us to calculate multistate structural ensembles that exhibit realistic spatial sampling and long-range correlations. Our initial studies were laborious and required a deep understanding of the underlying spin dynamics. Here, we present a MatLab package that integrates all data processing steps required to convert intensities of assigned peaks in NOESY series into upper and lower distance limits for structure calculation. Those steps include organization of the data in object format, extraction of autorelaxation and cross-relaxation rate constants by fitting of diagonal peak decays and cross peak buildups, validation of the data, correction for spin diffusion, graphical display of the results, and generation of distance limits in CYANA compatible format. The analysis may be carried out using a full relaxation matrix or a simplified "divide and conquer" approach that allows for partial deuteration of protons. As the program does not require expertise beyond that of standard resonance assignment/structure calculation, it is suitable for experts and nonexperts alike.
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| 9. |
- Sundell, Gustav, et al.
(författare)
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The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity : Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:1, s. 66-71
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Tidskriftsartikel (refereegranskat)abstract
- The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.
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| 10. |
- Vögeli, Beat, et al.
(författare)
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Enzyme Selectivity Fine-Tuned through Dynamic Control of a Loop.
- 2016
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 55:9, s. 3096-100
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Tidskriftsartikel (refereegranskat)abstract
- Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand-selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations.
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