Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY).

The prevailing understanding of various aspects of biochemical processes, including folding, stability, intermolecular interactions, and the binding of metals, substrates, and inhibitors, is derived from studies carried out under dilute and homogeneous conditions devoid of a crowding-related environment. The effect of crowding-induced modulation on the structure and stability of native and magnesium-dependent Chemotaxis Y (CheY), a bacterial signaling protein, was probed in the presence and absence of poly(ethylene glycol) (PEG). A combined analysis from circular dichroism, intrinsic and extrinsic fluorescence, and tryptophan fluorescence lifetime changes indicates that PEG perturbs the structure but leaves the thermal stability largely unchanged. Intriguingly, while the stability of the protein is enhanced in the presence of magnesium under dilute buffer conditions, PEG-induced crowding leads to reduced thermal stability in the presence of magnesium. Nuclear magnetic resonance (NMR) chemical shift perturbations and resonance broadening for a subset of residues indicate that PEG interacts specifically with a subset of hydrophilic and hydrophobic residues found predominantly in α helices, ß strands, and in the vicinity of the metal-binding region. Thus, PEG prompted conformational perturbation, presumably provides a different situation for magnesium interaction, thereby perturbing the magnesium-prompted stability. In summary, our results highlight the dominance of enthalpic contributions between PEG and CheY via both hydrophilic and hydrophobic interactions, which can subtly affect the conformation, modulating the metal-protein interaction and stability, implying that in the context of cellular situation, structure, stability, and magnesium binding thermodynamics of CheY may be different from those measured in dilute solution.

NMR investigations on binding and dynamics of imidazolium-based ionic liquids with HEWL.

Molecular level insights on protein-ionic liquid (P-IL) interactions are beneficial for assessing protein stability, binding and dynamics. In the present work, interactions of ILs, namely, 1-butyl 3-methylimidazolium methyl sulfate (IL1), 1-butyl 3-methylimidazolium octyl sulfate (IL2) and 1-butyl 3-methylimidazolium chloride (IL3) with hen egg white lysozyme (HEWL) protein were investigated using solution-state nuclear magnetic resonance (NMR) spectroscopy. To ascertain the binding and dynamics from the perspective of both protein and IL, various ligand based NMR approaches such as selective and non-selective nuclear spin-relaxation (R1SEL and R1NS), saturation transfer difference (STD), difference of inversion recovery rate with and without target irradiation (DIRECTION), 35Cl line-shape and spin-relaxation, and protein back bone amide chemical shift perturbations (CSPs) from 1H-15N HSQC were utilized. Among the ILs investigated, IL2 experiences significant interaction relative to those of IL1 and IL3, as revealed by the combined R1SEL and R1NS analysis, which is further supported by STD NMR. CSP analyses of 1H-15N HSQC spectra of aqueous P-IL mixtures enabled to identify the potential binding sites of ILs with HEWL. Whereas, 15N longitudinal (R1) and transverse (R2) spin-relaxation rates and 15N{1H} heteronuclear nuclear Overhauser effect (hetNOE) data subjected to the model free analysis for IL2 yielded the rotational correlation times and order parameters of various residues of HEWL. Furthermore, the results could discern the nature of interactions between studied ILs and HEWL in terms of specific and non-specific interactions.

Total Synthesis and Structural Revision of Monocillin VII.

The first asymmetric total synthesis of macrolactone monocillin VII and its C-10' epimer was achieved starting from a known chiral pure epoxide in 16 longest linear sequences. The present synthesis highlights the macrolactone formation involving an alkyne-dicobalt carbonyl complex under De Brabander's conditions followed by an unexpected regioselective hydration. The asymmetric total synthesis resulted in the revision of the configuration at C10' and reassignment of the absolute configuration of the natural product.

Residue-specific identification of phase separation hot spots of Alzheimer's-related protein tau.

Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient ß-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.

Simultaneous determination of fast and slow dynamics in molecules using extreme CPMG relaxation dispersion experiments.

Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr-Purcell-Meiboom-Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics. Using a fast folding protein, gpW, we have shown that previously inaccessible kinetics can be accessed with the improved precision and efficiency of the measurement by using this experiment.

High-power (1)H composite pulse decoupling provides artifact free exchange-mediated saturation transfer (EST) experiments.

Exchange-mediated saturation transfer (EST) provides critical information regarding dynamics of molecules. In typical applications EST is studied by either scanning a wide range of (15)N chemical shift offsets where the applied (15)N irradiation field strength is on the order of hundreds of Hertz or, scanning a narrow range of (15)N chemical shift offsets where the applied (15)N irradiation field-strength is on the order of tens of Hertz during the EST period. The (1)H decoupling during the EST delay is critical as incomplete decoupling causes broadening of the EST profile, which could possibly result in inaccuracies of the extracted kinetic parameters and transverse relaxation rates. Currently two different (1)H decoupling schemes have been employed, intermittently applied 180° pulses and composite-pulse-decoupling (CPD), for situations where a wide range, or narrow range of (15)N chemical shift offsets are scanned, respectively. We show that high-power CPD provides artifact free EST experiments, which can be universally implemented regardless of the offset range or irradiation field-strengths.

Speeding-up exchange-mediated saturation transfer experiments by Fourier transform.

Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R1ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states.

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments ­ (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH ­ which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins.

A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins.

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure-function studies.

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure-function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. (1)H and (13)C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment ((1)H(N), (15)N, (13)CO, (1)Hα/(13)Cα, and (1)Hß/(13)Cß chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca(2+) bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.

Parallel acquisition of 3D-HA(CA)NH and 3D-HACACO spectra.

We present here an NMR pulse sequence with 5 independent incrementable time delays within the frame of a 3-dimensional experiment, by incorporating polarization sharing and dual receiver concepts. This has been applied to directly record 3D-HA(CA)NH and 3D-HACACO spectra of proteins simultaneously using parallel detection of (1)H and (13)C nuclei. While both the experiments display intra-residue backbone correlations, the 3D-HA(CA)NH provides also sequential 'i - 1 → i' correlation along the (1)Hα dimension. Both the spectra contain special peak patterns at glycine locations which serve as check points during the sequential assignment process. The 3D-HACACO spectrum contains, in addition, information on prolines and side chains of residues having H-C-CO network (i.e., (1)Hß, (13)Cß and (13)COγ of Asp and Asn, and (1)Hγ, (13)Cγ and (13)COδ of Glu and Gln), which are generally absent in most conventional proton detected experiments.

Single point mutation induced alterations in the equilibrium structural transitions on the folding landscape of HIV-1 protease.

Equilibrium folding-unfolding transitions are hard to study in HIV-1 protease (PR) because of its autolytic properties. Further, the protease exhibits many tolerant point mutations some of which also impart drug resistance to the protein. It is conceivable that the mutations affect protein's function by altering its folding characteristics; these would clearly depend on the nature of the mutations themselves. In this background, we report here NMR studies on the effects of D25 N mutation, which removes one negative charge from the protein at the active site, on the equilibrium folding behaviour of PR starting from its acetic acid denatured state. It is observed that in PRD25N two slowly exchanging conformations are present at the N-terminal. One of them is similar to that of PR. Though the conformational and dynamics preferences of PR and PRD25N are fairly similar in 9 M acetic acid, they seem to undergo different folding transitions when acetic acid concentration is reduced. The differences are seen in the active site, in the flap, and in the hinge of the flap regions. The present study suggests that such differences, though different in detail, would occur for other mutations as well, and also for different initial denatured states. These would have significant regulatory implications for the efficacy of protease function.

Reduced dimensionality (4,3)D-HN(C)NH for rapid assignment of 1H(N)-15N HSQC peaks in proteins: an analytical tool for protein folding, proteomics, and drug discovery programs.

While nuclear magnetic resonance (NMR) has had commendable success in atomic-level investigation of folded proteins, intrinsically unfolded and partially folded proteins have always posed a great challenge, because of poor chemical shift dispersions. We present here a reduced-dimensionality-based NMR triple resonance pulse sequence, (4,3)D-HN(C)NH, which not only helps to disperse the peaks further by combining (15)N and amide (1)H chemical shifts, but also directly establishes correlations between (1)H(i)(N), (15)N(i), (1)H(i+1)(N), and (15)N(i+1) spins along the F(1)-F(3) planes. The F(2)-F(3) projection planes of this experiment provide unique identification of the check points in amide resonances. An assignment strategy derived by combining information along the F(1)-F(3) planes and in the F(2)-F(3) projection planes of the experiment has been presented and shown to be very useful for both intrinsically disordered/unfolded proteins and folded protein alike. The experiment and the protocol would be valuable for protein folding, proteomics, and drug discovery programs.

hnCOcaNH and hncoCANH pulse sequences for rapid and unambiguous backbone assignment in (13C, 15N) labeled proteins.

Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or (13)C' instead of (15)N during the t(1) period. Like HN(C)N, these spectra also exhibit special patterns of self and sequential peaks around glycines and prolines, which enable direct identification of certain triplets of residues and thus provide internal checks during the sequential assignment walk. The spectra enable rapid and unambiguous assignment of H(N), (15)N and (13)C(alpha) (or (13)C') in a single experiment, and thus would be of great value in high-throughput structural proteomics.