Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American eels (Anguilla rostrata).

Curcumin (Cur) exhibits diverse natural pharmacological activities, despite its limited water solubility (hydrophobicity) and low bioavailability. In this investigation, a valine-curcumin conjugate (Val-Cur) was synthesized through amino acid side chain modification, and its solubility increased to 1.78 mg/mL. In vitro experimental findings demonstrated that the antibacterial activity of Val-Cur against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus was significantly superior to that of Cur. The inhibition rate of Val-Cur against HepG2 (human hepatocellular carcinoma) cells was higher than that of Cur at low concentrations (below 25 µmol/L), although the IC50 value of Val-Cur did not differ significantly from that of Cur. In vivo biological effects of Val-Cur were assessed by adding it into the feed (150 mg/kg) of American eels (Anguilla rostrata). Val-Cur significantly improved the growth performance (↑weight gain rate, ↑specific growth rate, and ↓feed conversion rate) and activities of intestinal digestive enzymes (amylase and lipase) and antioxidant enzymes (superoxide dismutase) in American eels. Additionally, Val-Cur significantly improved serum biochemical indices (↑high-density lipoprotein cholesterol, ↓low-density lipoprotein cholesterol, ↓aspartate and alanine aminotransferases). Furthermore, Val-Cur increased intestinal microbial diversity, reduced the abundance of potentially pathogenic bacteria (Spiroplasma, Clostridium, and Pseudomonas), and elevated the abundance of beneficial digestion-promoting bacteria (Romboutsia, Phyllobacterium, Romboutsia sedimentorum, and Clostridium butyricum) conducive to glucose metabolism (P < 0.05). To the best of our knowledge, this study is the first to explore water-soluble curcumin in aquaculture, and the findings will lay the groundwork for the potential application of water-soluble curcumin in the field of aquaculture.

Molecular two-point recognition of fructosyl valine and fructosyl glycyl histidine in water by fluorescent Zn(II)-terpyridine complexes bearing boronic acids.

Selective recognition of fructosyl amino acids in water by arylboronic acid-based receptors is a central field of modern supramolecular chemistry that impacts biological and medicinal chemistry. Fructosyl valine (FV) and fructosyl glycyl histidine (FGH) occur as N-terminal moieties of human glycated hemoglobin; therefore, the molecular design of biomimetic receptors is an attractive, but very challenging goal. Herein, we report three novel cationic Zn-terpyridine complexes bearing a fluorescent N-quinolinium nucleus covalently linked to three different isomers of strongly acidified phenylboronic acids (ortho-, 2Zn; meta-, 3Zn and para-, 4Zn) for the optical recognition of FV, FGH and comparative analytes (D-fructose, Gly, Val and His) in pure water at physiological pH. The complexes were designed to act as fluorescent receptors using a cooperative action of boric acid and a metal chelate. Complex 3Zn was found to display the most acidic -B(OH)2 group (pKa = 6.98) and exceptionally tight affinity for FV (K = 1.43 × 105 M-1) with a strong quenching analytical response in the micromolar concentration range. The addition of fructose and the other amino acids only induced moderate optical changes. On the basis of several spectroscopic tools (1H, 11B NMR, UV-Vis, and fluorescence titrations), ESI mass spectrometry, X-ray crystal structure, and DFT calculations, the interaction mode between 3Zn and FV is proposed in a 1 : 1 model through a cooperative two-point recognition involving a sp3 boronate-diol esterification with simultaneous coordination bonding of the carboxylate group of Val to the Zn atom. Fluorescence quenching is attributed to a static complexation photoinduced electron transfer mechanism as evidenced by lifetime experiments. The addition of FGH to 3Zn notably enhanced its emission intensity with micromolar affinity, but with a lower apparent binding constant than that observed for FV. FGH interacts with 3Zn through boronate-diol complexation and coordination of the imidazole ring of His. DFT-optimized structures of complexes 3Zn-FV and 3Zn-FGH show a picture of binding which shows that the Zn-complex has a suitable (B⋯Zn) distance to the two-point recognition with these analytes. Molecular recognition of fructosyl amino acids by transition-metal-based receptors has not been explored until now.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet 13C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to 13C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13C-γ2-Ile and 13C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as 13C-δ1-Ile and 13C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13C methyl-labeled amino acids generated de novo by a versatile synthetic route.

Enantiodetermining processes in the synthesis of alanine, serine, and isovaline.

In this study, quantum chemical calculations were used to explore the synthesis of three chiral α-amino acids, specifically alanine, serine, and isovaline, from reactants found in interstellar space. Our focus is on the crucial step in the synthesis pathway that determines the chirality of the amino acids. The results indicate that in the case of alanine, the determination of enantiomer is primarily influenced by the direction of the collision of molecules or functional groups, which leads to the formation of a chirality center in a crucial intermediate. However, contrary to chemical expectations, the enantiodetermining/enantioselection step for serine and isovaline synthesis occurs prior to the creation of a chirality center.

Impact of non-proteinogenic amino acid norvaline and proteinogenic valine misincorporation on a secondary structure of a model peptide.

Norvaline is a straight-chain, hydrophobic, non-proteinogenic amino acid, isomeric with valine. Both amino acids can be misincorporated into proteins at isoleucine positions by isoleucyl-tRNA synthetase when the mechanisms of translation fidelity are impaired. Our previous study showed that the proteome-wide substitution of isoleucine with norvaline resulted in higher toxicity in comparison to the proteome-wide substitution of isoleucine with valine. Although mistranslated proteins/peptides are considered to have non-native structures responsible for their toxicity, the observed difference in protein stability between norvaline and valine misincorporation has not yet been fully understood. To examine the observed effect, we chose the model peptide with three isoleucines in the native structure, introduced selected amino acids at isoleucine positions and applied molecular dynamics simulations at different temperatures. The obtained results showed that norvaline has the highest destructive effect on the ß-sheet structure and suggested that the higher toxicity of norvaline over valine is predominantly due to the misincorporation within the ß-sheet secondary elements.

Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts: Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level.

Synthesis of fluorinated leucines, valines and alanines for use in protein NMR.

Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such as NaBD4, carbon-13C dioxide and sodium azide-1-15N. The lightly fluorinated leucines and valines were found to be good substrates for cell-free protein expression and even 3-fluoroalanine, which is highly toxic to bacteria in vivo, could be incorporated into proteins this way. 19F-NMR spectra of the protein GB1 produced with these amino acids showed large chemical shift dispersions. Particularly high incorporation yields and clean 19F-NMR spectra were obtained for GB1 produced with valine residues, which had been synthesized with a single fluorine substituting a hydrogen stereospecifically in one of the methyl groups.

The jasmonic acid-amino acid conjugates JA-Val and JA-Leu are involved in rice resistance to herbivores.

The phytohormone jasmonic acid (JA) plays a core role in plant defence against herbivores. When attacked by herbivores, JA and its bioactive derivatives are accumulated at the damage site, and subsequently perceived by the jasmonate co-receptors COI1 and JAZ proteins. The (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is known to be the main active JA derivative controlling vascular plant responses to herbivores as well as other JA-regulated processes. However, whether other endogenous JA-amino acid conjugates (JA-AAs) are involved in herbivore-induced defence responses remain unknown. Here, we investigated the role of herbivore-elicited JA-AAs in the crop plant rice. The levels of five JA-AAs were significantly increased under the armyworm, leaf folder and brown planthopper attack. Of the elicited JA derivatives, JA-Ile, JA-Val and JA-Leu could serve as ligands to promote the interaction between rice COI1 and JAZs, inducing OsJAZ4 degradation in vivo. JA-Val or JA-Leu treatment increased the expression of JA- and defence-related pathway genes but not JA-Ile levels, suggesting that these JA-AAs may directly function in JA signalling. Furthermore, the application of JA-Val or JA-Leu resulted in JA-mediated plant growth inhibition, while enhancing plant resistance to herbivore attack. This study uncovers that JA-Val and JA-Leu also play a role in rice defence against herbivores.

RIPK1 Is Cleaved by 3C Protease of Rhinovirus A and B Strains and Minor and Major Groups.

Rhinoviruses (RV), like many other viruses, modulate programmed cell death to their own advantage. The viral protease, 3C has an integral role in the modulation, and we have shown that RVA-16 3C protease cleaves Receptor-interacting protein kinase-1 (RIPK1), a key host factor that modulates various cell death and cell survival pathways. In the current study, we have investigated whether this cleavage is conserved across selected RV strains. RIPK1 was cleaved in cells infected with strains representing diversity across phylogenetic groups (A and B) and receptor usage (major and minor groups). The cleavage was abrogated in the presence of the specific 3C protease inhibitor, Rupintrivir. Interestingly, there appears to be involvement of another protease (maybe 2A protease) in RIPK1 cleavage in strains belonging to genotype B. Our data show that 3C protease from diverse RV strains cleaves RIPK1, highlighting the importance of the cleavage to the RV lifecycle.

Molecular Insight into the ß-Sheet Twist and Related Morphology of Self-Assembled Peptide Amphiphile Ribbons.

Self-assembly of high-aspect-ratio filaments containing ß-sheets has attracted much attention due to potential use in bioengineering and biomedicine. However, precisely predicting the assembled morphologies remains a grand challenge because of insufficient understanding of the self-assembly process. We employed an atomistic model to study the self-assembly of peptide amphiphiles (PAs) containing valine-glutamic acid (VE) dimeric repeats. By changing of the sequence length, the assembly morphology changes from flat ribbon to left-handed twisted ribbon, implying a relationship between ß-sheet twist and strength of interstrand hydrogen bonds. The calculations are used to quantify this relationship including both magnitude and sign of the ribbon twist angle. Interestingly, a change in chirality is observed when we introduce the RGD epitope into the C-terminal of VE repeats, suggesting arginine and glycine's role in suppressing right-handed ß-sheet formation. This study provides insight into the relationship between ß-sheet twist and self-assembled nanostructures including a possible design rule for PA self-assembly.

RP-HPLC method development and validation for quantification of daclatasvir dihydrochloride and its application to pharmaceutical dosage form.

Daclatasvir dihydrochloride is an antiviral drug used in the treatment of Hepatitis C and for its estimation in drug product, no Pharmacopeial method is available. Therefore, a simple, rapid, precise and accurate isocratic RP-HPLC method was developed and validated for quantification of daclatasvir dihydrochloride in pharmaceutical dosage form. The quantification was carried out using Hypersil ODS - C18 Column (250mm, 4.6mm, 5µm), Shimadzu LC-2030 Prominence-I Series. The mobile phase composed of phosphate buffer (pH 3.5, adjusted with ortho phosphoric acid) and acetonitrile (60:40 v/v). The flow rate was 1.0ml/min with UV detection at 308 nm. The validation of developed method was conducted for specificity, linearity, accuracy, precision, LOD and LOQ. A linearity was established in the concentration range of 0.5-150% with coefficient of correlation 0.9993. The limit of detection (LOD) was 0.005µg/ml and the limit of quantification (LOQ) was 0.01µg/ml. The method was successfully applied to the assay and in-vitro dissolution studies of daclatasvir dihydrochloride in tablet dosage form. It can be concluded that this method can be very helpful in the quality control estimation of daclatasvir dihydrochloride in different pharmaceutical products intended for hepatitis C infections.

Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode.

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Determination of modification degree of polysialylated therapeutic proteins using 1H-NMR spectroscopy.

Water soluble polymers and their derivatives bound to proteins can dramatically favor the biological activity of new drugs and vaccines. Quantification of the modification degree of the protein is crucial during the development and licensing phase and later in order to monitor the industrial production process and to match product specification. In this work, we describe an innovative way to measure directly the modification degree of polysialylated proteins using proton NMR (Nuclear Magnetic Resonance) spectroscopy. Following a calibration step, the modification degree can be easily deduced by the integration ratio of a separate signal from the polymer and selected signals from the protein. In fact, the upfield-shifted signals of methyl groups from Valine, Leucine and Isoleucine can be used as an internal calibration reference for the integration. In this paper recombinant factor VIII (rFVIII) and recombinant factor IX (rFIX) proteins modified by polysialic acid (PSA) are used to illustrate the accuracy, reproducibility and ease of the method that may replace or complement wet-chemistry approaches.

Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence of L-tryptophan.

The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand L-tryptophan (L-Trp) indicate that L-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with L-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of L-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis.

V367F Mutation in SARS-CoV-2 Spike RBD Emerging during the Early Transmission Phase Enhances Viral Infectivity through Increased Human ACE2 Receptor Binding Affinity.

The current pandemic of COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, we first analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human angiotensin-converting enzyme 2 (ACE2) receptor. Among 32,123 genomes of SARS-CoV-2 isolates (December 2019 through March 2020), 302 nonsynonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F+D614G), which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. IMPORTANCE A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious has been the research hot spot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human angiotensin-converting enzyme 2 (ACE2) receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.

Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain.

Methyl moieties are highly valuable probes for quantitative NMR studies of large proteins. Hence, their assignment is of the utmost interest to obtain information on both interactions and dynamics of proteins in solution. Here, we present the synthesis of a new precursor that allows connection of leucine and valine pro-S methyl moieties to backbone atoms by linear 13C-chains. This optimized 2H/13C-labelled acetolactate precursor can be combined with existing 13C/2H-alanine and isoleucine precursors in order to directly transfer backbone assignment to the corresponding methyl groups. Using this simple approach leucine and valine pro-S methyl groups can be assigned using a single sample without requiring correction of 1H/2H isotopic shifts on 13C resonances. The approach was demonstrated on the N-terminal domain of human HSP90, for which complete assignment of Ala-ß, Ile-δ1, Leu-δ2, Met-ε, Thr-γ and Val-γ2 methyl groups was obtained.