| 1. |
- Akke, Mikael, et al.
(författare)
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Protein conformational dynamics by relaxation dispersion
- 2013
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Ingår i: Encyclopedia of biophysics. - Berlin : Elsevier. - 9783642167126 ; , s. 1967-1979
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Protein dynamics is intimately connected to function. Many biochemical reactions are mediated by protein conformational transitions to high-energy states that are often populated at low levels undetectable by traditional methods in structural biology. Nuclear magnetic resonance ( NMR) relaxation dispersion methods ( Relaxation Dispersion) provide a unique means of characterizing this type of protein motion with rate constants ranging from approximately 1–105 s–1 and populations down to approximately 1%. Relaxation dispersion experiments yield a wealth of information about the exchanging system, including the kinetic rate constants, the relative populations of the exchanging states, and the chemical shift differences between these, which depend on the molecular structure. Temperature-dependent experiments enable mapping of the energy landscape in terms of the free energy barrier and the free energy difference between the exchanging states, broken down into enthalpy and entropy. Applications have addressed protein folding-unfolding ( Protein Folding), enzyme catalysis ( Protein Dynamics in Catalysis – Computational Studies), and ligand binding ( Protein–Ligand Dynamics) (Korzhnev and Kay 2008; Baldwin and Kay 2009). This chapter outlines the fundamental principles of relaxation dispersion experiments and their application to protein dynamics. The focus is on heteronuclear experiments performed on isotope-labeled proteins ( Protein NMR – Introduction), where the dynamics is typically probed by 15N or 13C spin relaxation, but experiments have also been developed for 1H.
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| 2. |
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| 3. |
- Björnsson, Jon Mar, et al.
(författare)
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Reduced proliferative capacity of hematopoietic stem cells deficient in hoxb3 and hoxb4
- 2003
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Ingår i: Blood. - 0006-4971 .- 1528-0020. ; 23:11, s. 3872-3883
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Tidskriftsartikel (refereegranskat)abstract
- Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin(-) Scal(+) c-kit(+) hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.
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| 4. |
- Cala, Juan, et al.
(författare)
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Graded modules over object-unital groupoid graded rings
- 2021
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Ingår i: Communications in Algebra. - : Taylor & Francis Group. - 0092-7872 .- 1532-4125. ; 50:2, s. 444-462
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Tidskriftsartikel (refereegranskat)abstract
- In this article, we analyze the category(Formula presented) of unitary G-graded modules over object unital G -graded rings R, being G a groupoid. Here we consider the forgetful functor G - R- mod and determine many properties (Formula presented.) for which the following implications are valid for modules M in (Formula presented.) M is (Formula presented.) (Formula presented.) U(M) is (Formula presented.) U(M) is (Formula presented.) (Formula presented.) M is (Formula presented.) We treat the cases when (Formula presented.) is any of the properties: direct summand, projective, injective, free and semisimple. Moreover, graded versions of results concerning classical module theory are established, as well as some structural properties (Formula presented.).
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| 5. |
- Cala, Juan, et al.
(författare)
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Object-unital groupoid graded rings, crossed products and separability
- 2021
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Ingår i: Communications in Algebra. - : Informa UK Limited. - 0092-7872 .- 1532-4125. ; 44:4, s. 1676-1696
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Tidskriftsartikel (refereegranskat)abstract
- We extend the classical construction by Noether of crossed product algebras, defined by finite Galois field extensions, to cover the case of separable (but not necessarily finite or normal) field extensions. This leads us naturally to consider non-unital groupoid graded rings of a particular type that we call object unital. We determine when such rings are strongly graded, crossed products, skew groupoid rings and twisted groupoid rings. We also obtain necessary and sufficient criteria for when object unital groupoid graded rings are separable over their principal component, thereby generalizing previous results from the unital case to a non-unital situation. © 2020 The Author(s). Published with license by Taylor and Francis Group, LLC.
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| 6. |
- Chi, Celestine N., et al.
(författare)
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Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:44, s. 8971-8979
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Tidskriftsartikel (refereegranskat)abstract
- The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.
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| 7. |
- 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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| 8. |
- Korzhnev, Dmitry M., et al.
(författare)
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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
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 372:2, s. 497-512
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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.
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| 9. |
- Lundström, Patrik, 1971-, et al.
(författare)
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A single-quantum methyl C-13-relaxation dispersion experiment with improved sensitivity
- 2007
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Ingår i: Journal of Biomolecular NMR. - : Springer. - 0925-2738 .- 1573-5001. ; 38:1, s. 79-88
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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.
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| 10. |
- Lundström, Patrik, 1971-, et al.
(författare)
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Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:5, s. 1915-1926
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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.
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