Accurately Probing Slow Motions on Millisecond Timescales with a Robust NMR Relaxation Experiment

A new pulse scheme is proposed for the accurate measurement of relaxation dispersion, which cycles the phases of CPMG pulses. Numerical simulations show that systematic errors in the measured relaxation rates mainly result from off-resonance and radio frequency inhomogeneity effects and they can be significantly suppressed with the method proposed here. The method has been demonstrated on human liver fatty acid binding protein. It allows the reliable identification of residues undergoing conformational exchange on millisecond timescales and accurate extraction of kinetics parameters. The relaxation dispersion data indicate that human liver fatty acid binding protein is highly flexible on millisecond timescales

Impact of the α‑Synuclein Initial Ensemble Structure on Fibrillation Pathways and Kinetics

The presence of intracellular filamentous α-synuclein (αS) aggregates is a common feature in Parkinson’s disease. Recombinant expressed and purified human αS is also capable of forming fibrils in vitro. Many studies have shown that solution conditions heavily influence αS fibrillation kinetics, fibril structure, and morphology that exhibit differential biological effects. Nevertheless, the αS ensemble structure in various solution conditions is not well characterized; furthermore, how the initial solution ensemble structures impact αS assembly kinetics and pathways that result in diverse fibril structure and morphology remains elusive. Here, we mainly employed NMR spectroscopy to characterize the initial ensemble structure of αS in the presence or absence of a 150 mM sodium chloride (NaCl) solution, where two polymorphs of αS were demonstrated in previous studies. Our data show that αS exhibits distinct conformations and fibrillation kinetics in these two solutions. αS adopts a more compact and rigid ensemble structure that has faster fibrillation kinetics in the absence of NaCl. On the basis of the ensemble structure and dynamics, we proposed a possible molecular mechanism in which αS forms different polymorphs under these two conditions. Our results provide novel insights into how the initial conformation impacts fibrillation pathways and kinetics, suggesting that a microenvironment can be used to regulate the intrinsically disordered proteins assembly

Additional file 1: Figure S1. of α-synuclein-lanthanide metal ions interaction: binding sites, conformation and fibrillation

Chemical shifts and intensities changes of amide groups in αS at various concentration of Lu3+. Figure S2. Tb3+ effects on 1D 1H spectra of αS aromatic side chains. Figure S3. Dy3+ effects on 1D 1H spectra of αS aromatic side chains. Figure S4. Ca2+ effects on αS 1H-15N-HSQC spectra. Figure S5. Chemical shifts and intensities changes of amide groups in αS at various concentration of Ca2+. Figure S6. Al3+ effects on αS 1H-15N-HSQC spectra. Figure S7. Intensities changes of amide groups in αS at various concentration of Al3+. Figure S8. Fibrillation of α-synuclein monitored by ThT fluorescence in the presence of different lanthanide metal ions. (DOC 1028 kb

Proton NMR Based Investigation of the Effects of Temperature and NaCl on Micellar Properties of CHAPS

The effects of temperature and NaCl on the micellization of CHAPS, a zwitterionic detergent widely used in membrane protein studies, have been investigated by NMR spectroscopy. We found that the two apparent critical micelle concentration (cmc) values of CHAPS decrease with the increase of temperature, as well as the NaCl concentration. The thermodynamic parameters derived from the temperature-dependent cmc values show that the micellization process is spontaneous and exothermic, and the van der Waals interaction is likely to be the main factor for the micellization of CHAPS. The micellar hydrodynamic radii remain almost the same in a range of 100−600 mM NaCl, indicating that the aggregate states of CHAPS are not sensitive to the change of the surrounding conditions. In addition, the dependence of nuclear Overhauser effect (NOE) intensities on temperatures further demonstrates the existence of the unique staggered micellar structure of CHAPS at a concentration above the apparent second cmc, which was suggested in our previous work. Our results provide a basis for optimizing CHAPS concentration in the solubilization or stabilization of membrane proteins under nondenaturing conditions and may be helpful to understand its interaction with proteins

Concentration-Dependent Aggregation of CHAPS Investigated by NMR Spectroscopy

CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is a zwitterionic surfactant, which has been extensively used in various biological fields. In the present work, the concentration-dependent aggregation of the surfactant in deuterium oxide solution was investigated by NMR spectroscopy. We have found that two break points exist on the basis of the NMR parameters such as chemical shift difference (Δδ), self-diffusion coefficient (D), and relaxation rates (R1, R2). The first break point corresponds to the widely accepted normal critical micelle concentration (cmc). The second one is unexpected and ascribed to the second cmc, indicating that there is another type of micelle at higher concentrations. Further analysis using 1D selective NOESY with spin-diffusion quenching reveals that at the concentration above the second cmc CHAPS may form a two-layer spherical structure of micelles, with the aliphatic groups of CHAPS molecules in the inner layer interact with the steroid groups in the outer layer. The existence of two types of micelles has also been supported by our TEM experiment. The dependence of CHAPS micelle size on concentration explains why some proteins are soluble and stable only at concentrations above the second cmc. Therefore, our finding provides a basis for optimizing CHAPS concentration in functional and structural studies of membrane proteins

Beryllium Fluoride Exchange Rate Accelerated by Mg²⁺ as Discovered by ¹⁹F NMR

Beryllium fluoride is widely used as a phosphoryl analogue in macromolecular studies, which are not only fluoride-sensitive but also magnesium-dependent. The beryllium fluorides are a mixture of different species including BeF₃^– and BeF₄^2– exchanging under thermodynamic equilibrium in neutral aqueous solutions. In the cases of mimicking phosphate group transfer, both beryllium fluoride and the magnesium ion are generally needed. However, the impact of magnesium on the bioactivity of beryllium fluoride is not clear. We have found by ¹⁹F NMR spectroscopy that Mg²⁺ can severely affect the chemical exchange kinetics between BeF₃^– and BeF₄^2–. When the F^– concentration is relatively low, the presence of 10.0 mM Mg²⁺ can accelerate the exchange rate 3–4 fold. However, when the F^– concentration is relatively high, the Mg²⁺ effect on the chemical exchange vanishes. On the basis of these findings, we proposed a possible mechanism that BeF₄^2– and Mg²⁺ form an ion pair that affects the distribution of beryllium fluoride species and thus the activity in the solution

Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB from Escherichia coli

PhoB is a response regulator of PhoR/PhoB two-component system from Escherichia coli that is involved in the environmental phosphate regulation. It has been reported that the N-terminal receiver domain (PhoBN) forms a dimer using the α1−α5 face in the apo state and a dimer using the α4−β5−α5 face in the active state investigated by X-ray crystallography. However, it is not clear whether the conformational switch of the dimer is dependent on phosphorylation. Here, we report the NMR studies of PhoBN in solution in its apo form. We observed that the secondary structural fragments of apo PhoBN characterized by NMR are almost the same as those determined by crystallography, but the NMR spectrum of PhoBN shows inhomogeneous amide signals. Concentration dilution experiments and backbone relaxation parameters showed that PhoBN exists in equilibrium between monomer and dimer states. Using paramagnetic relaxation enhancement experiments, we demonstrated that the dimer of apo PhoBN forms several transient dimer interfaces in solution. This finding suggested that, in addition to the monomer-to-dimer exchange, the inactive conformation of PhoBN has different domain arrangements, which are independent of phosphorylation. It provides an experimental data for the conformational selection mechanism of the phosphorylation of PhoBN

Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity

Pin1 is a two-domain peptidyl–prolyl isomerase (PPIase) associated with neurodegeneration and tumorigenesis. The two domains, a WW and a PPIase domain, are connected by a flexible linker, making Pin1 adopt various conformations ranging from compact to extended, wherein Pin1 exhibits different extents of interdomain contact. Previous studies have shown that weakening interdomain contact increases the isomerase activity of Pin1. Here, we propose an NMR chemical shift correlation-analysis-based method that will be general for two-domain proteins to gauge two-state populations of Pin1, and we report a linker-modified mutant of Pin1 with enhanced interdomain contact and increased isomerase activity, with the latter suggesting an uncorrelated effect of interdomain contact on isomerase activity. Thus, although bindings of different substrates in the WW domain impose opposite effects on interdomain contact, in both cases, it may promote isomerization, implying cooperativity between substrate binding in the WW domain and isomerization in the PPIase domain

Creating Conformational Entropy by Increasing Interdomain Mobility in Ligand Binding Regulation: A Revisit to N-Terminal Tandem PDZ Domains of PSD-95

The two N-terminal PDZ domains of postsynaptic density protein-95 (PDS-95 PDZ1 and PDZ2) are closely connected in tandem by a conserved peptide linker of five amino acids. The interdomain orientation between PDZ1 and PDZ2 of the ligand-free PDZ12 tandem is restrained, and this conformational arrangement facilitates the synergistic binding of PDZ12 to multimeric targets. The interdomain orientation of the target-bound state of PDZ12 is not known. Here, we have solved the structure of PDZ12 in complex with its binding domain from cypin. Both chemical shift data and residual dipolar coupling measurements showed that the restrained interdomain orientation disappeared upon cypin peptide binding. NMR-based relaxation experiments revealed slow interdomain motions in the PDZ12/cypin peptide complex. Molecular dynamics simulations also showed that the PDZ12/cypin complex has larger conformational flexibility than the ligand-free PDZ12. This dramatic change of protein dynamics provides extra conformational entropy upon ligand binding, thus enhancing the ligand binding affinity of the PDZ12 tandem. Modulation of ligand binding affinity through concerted interdomain structural and dynamic rearrangements may represent a general property of multidomain scaffold proteins

A Selective NMR Method for Detecting Choline Containing Compounds in Liver Tissue: The ¹H−¹⁴N HSQC Experiment

The feasibility of a 1H−14N HSQC experiment on tissues is demonstrated with a mouse liver based on the J couplings between the protons and the quadrupolar nucleus 14N in choline. Free choline, phosphocholine, and glycerolphosphocholine 1H−14N HSQC signals were selectively observed with all unwanted signals cleanly suppressed. The CH2O signals were well resolved in the two-dimensional spectrum, which can be used for quantitative analyses