CPMG sequences with enhanced sensitivity to chemical exchange

Improved relaxation-compensated Carr–Purcell–Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone ¹⁵N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc. 121, 2331–2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from ¹H-¹H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional ¹⁵N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor.     

The use of TROSY for detection and suppression of conformational exchange NMR line broadening in biological macromolecules

The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled ¹H and ¹⁵N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [¹⁵N,¹H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base ¹H–¹⁵N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D ^h1 J _HN,^h2 J _NN-quantitative [¹⁵N,¹H]-TROSY to measure ^h1 J _HN and ^h2 J _NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (^h2 J _NN+^h1 J _NH)-correlation-[¹⁵N,¹H]-TROSY to correlate ¹H^N chemical shifts of bases with the chemical shifts of the tertiary ¹⁵N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids.     

TROSY-selected ZZ-exchange experiment for characterizing slow chemical exchange in large proteins

A TROSY-selected ZZ-exchange experiment is described for measuring slow chemical exchange rates by monitoring the TROSY component of ¹⁵N longitudinal magnetization. Application of the proposed pulse sequence to the cadherin 8 N-terminal extracelluar domain demonstrates that enhanced sensitivity is obtained, compared to a previously described TROSY-detected ZZ-exchange sequence (Sahu et al. J Am Chem Soc 129: 13232–13237, 2007), by preserving the TROSY effect during the mixing period as well as the frequency encoding periods.     

Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach

Relaxation compensated constant-time Carr–Purcell–Meiboom–Gill relaxation dispersion experiments for amide protons are presented that detect s-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180° pulses can be applied to ¹H than to ¹⁵N spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of ¹H experiments. Applications to [¹H-¹⁵N]-ubiquitin and [¹H-¹⁵N]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear ³J(H_N-H) coupling. In the case of the protease, we show that relaxation dispersion of both ¹H and ¹⁵N spins provides a more comprehensive picture of slow backbone dynamics than does the relaxation dispersion of either spin alone. We also compare the relative merits of ¹H versus ¹⁵N transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types.     

Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry

Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.     

A new approach for obtaining sequential assignment of large proteins

A novel NMR experiment for obtaining sequential assignment of large proteins and protein complexes is described. The proposed method takes full advantage of transverse relaxation optimized spectroscopy (TROSY) and utilizes spin-state-selection to distinguish between intraresidual and sequential connectivities in the HNCA-TROSY-type correlation experiment. Thus, the intra- and interresidual cross peaks can be identified without relaying magnetization via carbonyl carbon, which relaxes very rapidly at the high magnetic fields where TROSY is most efficient. In addition, the presented method enables measurement of several scalar and residual dipolar couplings, which can potentially be used for structure determination of large proteins.     

Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution

Chemical (conformational) exchange on the ms-s time scale is reliably identified by the observation of transverse relaxation rates, R_ex, that depend upon the strength of the effective field (_1eff=B_1eff) used in spin lock or CPMG experiments. In order to determine if the exchange correlation time, _ex, is the fast or slow limit, measurements of (i) signal line shape and (ii) temperature dependence of R_ex have been commonly used in studies of stable, small molecules. However, these approaches are often not applicable to proteins, because sample stability and solubility, respectively, limit the temperature range and signal sensitivity of experiments. Herein we use a complex, but general, two-site exchange equation to show when the simple fast exchange equations for R_ex are good approximations, in the case of proteins. We then present a simple empirical equation that approximately predicts R_ex in all exchange regimes, and explains these results in a clear, straightforward manner. Finally we show how one can reliably determine whether _ex is in the fast or slow exchange limit.     

A guide to the effects of a large portion of the residues of triosephosphate isomerase on catalysis,stability, druggability,and human disease

Triosephosphate isomerase (TIM) is a ubiquitous enzyme, which appeared early in evolution. TIM is responsible for obtaining net ATP from glycolysis and producing an extra pyruvate molecule for each glucose molecule, under aerobic and anaerobic conditions. It is placed in a metabolic crossroad that allows a quick balance of the triose phosphate aldolase produced by glycolysis, and is also linked to lipid metabolism through the alternation of glycerol‐3‐phosphate and the pentose cycle. TIM is one of the most studied enzymes with more than 199 structures deposited in the PDB. The interest for this enzyme stems from the fact that it is involved in glycolysis, but also in aging, human diseases and metabolism. TIM has been a target in the search for chemical compounds against infectious diseases and is a model to study catalytic features. Until February 2017, 62% of all residues of the protein have been studied by mutagenesis and/or using other approaches. Here, we present a detailed and comprehensive recompilation of the reported effects on TIM catalysis, stability, druggability and human disease produced by each of the amino acids studied, contributing to a better understanding of the properties of this fundamental protein. The information reviewed here shows that the role of the noncatalytic residues depend on their molecular context, the delicate balance between the short and long‐range interactions in concerted action determining the properties of the protein. Each protein should be regarded as a unique entity that has evolved to be functional in the organism to which it belongs. Proteins 2017; 85:1190–1211. © 2017 Wiley Periodicals, Inc.     

Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous ¹H–¹⁵N/¹³CH₃ NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both ¹H–¹⁵N and methyl ¹³CH₃ spin-systems in the course of indirect evolution (t ₂) and acquisition period (t ₃) of 3D NOESY experiments. The methodology has been tested on {U-[¹⁵N,²H]; Ileδ1-[¹³CH₃]; Leu,Val-[¹³CH₃,¹²CD₃]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.     

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R_1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide ¹H off-resonance R_1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using ¹⁵N or ¹³C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The ¹H off-resonance R_1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for ¹⁵N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly ¹⁵N-enriched samples that were either highly enriched in ²H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the ¹H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present ¹H off-resonance R_1ρ experiments are in excellent agreement with those determined previously using a combination of ¹⁵N laboratory-frame and off-resonance R_1ρ relaxation data, with average values of and 21 ± 3 μs, respectively.     

Using chemical exchange to assign non-covalent protein complexes in slow exchange with the free state: Enhanced resolution and efficient signal editing

The formation of a ligand-protein complex oftentimes results in significant chemical shift changes. These changes may occur not only in the binding pocket but also in distal regions of the protein target. Therefore the reassignment of the backbone resonances in the complex is frequently a time consuming challenge. Here we present a suite of resolution-enhanced N(z)-exchange NMR experiments useful for rapidly assigning backbone (1)H and (15)N amide resonances of the ligand-bound form of a protein in slow exchange with its free state. Incorporation of semi-constant time frequency labeling periods into 3D N(z)-exchange experiments in combination with the collection of resolution-enhanced 2D N(z)-exchange difference spectra leads to a powerful set of tools for analyzing protein-ligand complexes. This allows for both the assignment of the bound state and the rapid assessment of the protein binding interface. The proposed methodology is demonstrated on the complex formed by the dimerization-docking domain of the c-AMP-dependent protein kinase and the tethering domain of the dual-binding A-kinase anchoring protein (AKAP).     

Improved HSQC experiments for the observation of exchange broadened signals

Summary We present here HSQC experiments with improved sensitivity for signals in the presence of exchange broadening. During periods of coherence transfer through scalar coupling the experiments employ CPMG-derived pulse trains to reduce loss of dephasing of spin coherence due to chemical exchange. ¹⁵N–¹H gradient CPMG-HSQC and SE-CPMG-HSQC experiments have been developed and applied to complexes of lac repressor headpiece with operator DNA. Improved sensitivity is demonstrated for many protein backbone and side-chain resonances in the complex, markedly for signals of protons located at the protein-DNA interface. In addition, a significant increase in intensity is observed for arginine guanidino groups undergoing conformational exchange.     

A statistical mechanical model for hydrogen exchange in globular proteins.

We develop a statistical mechanical theory for the mechanism of hydrogen exchange in globular proteins. Using the HP lattice model, we explore how the solvent accessibilities of chain monomers vary as proteins fluctuate from their stable native conformations. The model explains why hydrogen exchange appears to involve two mechanisms under different conditions of protein stability; (1) a “global unfolding” mechanism by which all protons exchange at a similar rate, approaching that of the denatured protein, and (2) a “stable-state” mechanism by which protons exchange at rates that can differ by many orders of magnitude. There has been some controversy about the stable-state mechanism: does exchange take place inside the protein by solvent penetration, or outside the protein by the local unfolding of a subregion? The present model indicates that the stable-state mechanism of exchange occurs through an ensemble of conformations, some of which may bear very little resemblance to the native structure. Although most fluctuations are small-amplitude motions involving solvent penetration or local unfolding, other fluctuations (the conformational distant relatives) can involve much larger transient excursions to completely different chain folds.     

A study of protein-water exchange through the off-resonance ROESY experiment: Application to the DNA-binding domain of AlcR

Summary In this communication a new NMR experiment for the safe observation and quantification of water-protein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shift-based separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.     

A TROSY relayed HCCH-COSY experiment for correlating adenine H2/H8 resonances in uniformly 13C-labeled RNA molecules

A new TROSY relayed HCCH-COSY pulse sequence is introduced for correlating adenine H2 and H8 resonances in ¹³C-labeled RNA molecules. The pulse scheme provides substantial improvements in signal-to-noise compared to previously suggested experiments, and therefore will be suitable for NMR studies of larger RNA molecules. The experiment provides ¹³C chemical shifts for all carbon nuclei in the adenine base. This is advantageous for resolving spectral overlap in larger RNA molecules and provides a starting point for measuring additional parameters for these carbons in the adenine spin system.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.