Clean SEA-HSQC: a method to map solvent exposed amides in large non-deuterated proteins with gradient-enhanced HSQC

The recent introduction of the SEA-TROSY experiment (Pellecchia et al. (2001) J. Am. Chem. Soc., 123, 4633–4634) can alleviate the problem of resonance overlap in ¹⁵N/²H labeled proteins. This method selectively observes solvent exposed amide protons with a SEA element. However, SEA-TROSY spectra may be contaminated with exchange-relayed NOE contributions from fast exchanging hydroxyl or amine protons and longitudinal relaxation contributions. Furthermore, for non-deuterated proteins or protein-ligand complexes, SEA-TROSY spectra may contain NOE contributions from aliphatic protons. In this communication, a modified version of the SEA element, a Clean SEA element, is introduced to eliminate these artifacts.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Mapping of the detergent-exposed surface of membrane proteins and peptides by 1H solution NMR in detergent: Application to the gramicidin A ion channel

The present work evaluates the use of intermolecular polypeptide–detergent 1H through-space connectivities to determine the bilayer exposed-surface and the bilayer topography of membrane polypeptides solubilized in non- deuterated detergents. For this purpose, the membrane peptide gramicidin A, solubilized in non-deuterated sodium dodecylsulfate as its dimeric 6,3 helix channel conformation was used. For this peptide, a high-resolution 3D structure, as well as reasonable assumptions concerning its membrane arrangement, exist. Band-selective 2D NOESY, ROESY and 3D NOESY-NOESY experiments were used to detect detergent–polypeptide through-space correlations in the presence of an excess of the non-deuterated detergent. The observed intermolecular NOEs appear to be strongly temperature- dependent. Based on the known 3D structure of the gramicidin channel, the detergent–polypeptide through-space correlations appear to be selective for 1H located on the hydrophobic surface of gramicidin A with very few contributions from interior 1H or water-exposed 1H. It is suggested that this method can be of general use to evaluate the bilayer-exposed surface and topography of membrane peptides and small proteins.     

NMR studies of aurein 1.2 analogs

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA > aurein 1.2 >> the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by ¹H NMR spectroscopy, including structural refinement by natural abundance ¹³C^α, ¹³C^β, and ¹⁵N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 Å below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly ²H, ¹³C, ¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

Combined solid state and solution NMR studies of α,ɛ-<Superscript>15</Superscript>N labeled bovine rhodopsin

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of α,ɛ-¹⁵N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state ¹³C,¹⁵N-REDOR and HETCOR experiments of all possible ¹³C′ _i-1 carbonyl/¹⁵N _i -tryptophan isotope labeled amide pairs, and H/D exchange ¹H,¹⁵N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone ¹⁵N nuclei and partially to their bound protons. ¹H,¹⁵N chemical shift assignment was achieved for indole side chains of Trp35^1.30 and Trp175^4.65. ¹⁵N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175^4.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.     

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

Abstract

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic α-helical structure from Lys² to Leu¹⁴. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp⁹ are in close contact with methylene protons of SDS micelles. T₁ relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp⁹ with Ala⁹, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp⁹ into the membrane, as well as the amphiphilic α-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Isotope-edited multidimensional NMR of calcineurin B in the presence of the non-deuterated detergent CHAPS

Summary At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to ¹⁵N or ¹³C are removed by isotope filtering and purge pulses.     

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with ¹⁵N. The requirement of ¹³C-/¹⁵N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., _0c~1). The technique was applied to an unstructured ¹⁵N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s^-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.     

Detergent binding to unmyristylated protein kinase A—Structural implications for the role of Myristate

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60^v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knightonet al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlssonet al., 1993; Zhenget al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zhenget al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This inside out motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60^v-src.     

Residues of Aliphatic and Polycyclic Aromatic Hydrocarbons in Some Fish Species of Lake Temsah,Ismailia, Egypt: An Analytical Search for Hydrocarbon Sources and Exposure Bioindicators

Residues of aliphatic and polycyclic aromatic hydrocarbons (PAHs) were monitored in some fish species collected from Temsah Lake, near Ismailia, Egypt. Fish were selected to represent different feeding habits and ecological niches in the lake's ecosystem. Fish samples were extracted using organic solvents, and residues of aliphatic and PAHs were separated using column chromatography and detected using gas liquid chromatography. Fish species were Clupea sirm, Mugil sehli, Mugil capito, Morone labrax, and Sciasna sp. Clupea sirm, a surface feeder fish had the highest concentration of aliphatic hydrocarbons, 320 ± 54 ng g^?1, while Morone labrax, a predatory fish that live in the water column, had the highest concentration of PAHs, 315.87 ± 46 ng g^?1. Even-number aliphatic hydrocarbons were more frequently detected in all fish species in comparison to odd-number aliphatic hydrocarbons, suggesting a petrogenic origin of these compounds. Meanwhile, the pattern of PAHs detected in the present study suggested that they originate from atmospheric deposition rather than land-based runoff. Morone labrax fish and Clupea sirm fish were the most suitable candidate bioindicators of exposure to aliphatic hydrocarbons and PAHs through fish consumption of the five fish species examined in this study.     

Development of ETB Selective Agonists: Solution Structure of a Linear Endothelin-1 Analogue,ET-1 [Cys(Acm)1,15, Ala3, Leu7, dAsp8, Aib11]

Abstract

The solution structure of a synthetic ET_B selective agonist, ET-l[Cys(Acm)^1,15, Ala³, Leu⁷, dAsp⁸, Aib¹¹] has been solved by ¹H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an α-helical conformation between residues Leu⁶-His¹⁶ and by N- and C-termi- ni which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ETB selective agonists, are proving to be important in the understanding of how they interact with the ETB receptor.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

Interactions between ¹⁵N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in ¹³C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C^α signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the ³¹P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The ¹³C-peptide peaks were selectively detected in a ¹³C-detected ¹H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The ¹³C-MAOSS results thus independently confirms previous findings from ¹⁵N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O’Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428–9437]. In summary, application of house hold foil opens the possibility of measuring high resolution ¹³C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates.     

Selective Reconstitution of the Tonoplast H+-ATPase of the Crassulacean-Acid Metabolism Plant Kalanchoë daigremontiana

IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoë daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H⁺-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H⁺-ATPase.     

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in ¹⁵N or ¹³C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in ¹H–¹⁵N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.     

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives and determination of their inhibition properties against human carbonic anhydrase I and II isoenzymes

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acid (4) derivatives containing structural characteristics that can be used for the synthesis of several active molecules, is presented. Some of the butenoic acid derivatives (4a, 4c, 4e, 4i, 4j, 4k) are synthesized following literature procedures and at the end of the reaction. In addition, structures of all synthesized derivatives (4a–4m) were determined by ¹H-NMR, ¹³C-NMR and IR spectroscopy. Carbonic anhydrase is a metalloenzyme involved in many crucial physiologic processes as it catalyzes a simple but fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Significant results were obtained by evaluating the enzyme inhibitory activities of these derivatives against human carbonic anhydrase hCA I and II isoenzymes (hCA I and II). Butenoic acid derivatives (4a–4m) strongly inhibited hCA I and II with K_is in the low nanomolar range of 1.85?±?0.58 to 5.04?±?1.46?nM against hCA I and in the range of 2.01?±?0.52 to 2.94?±?1.31?nM against hCA II.