2D 1H Nuclear Magnetic Resonance Research Articles

Evaluation of the Metabolic Profile of Arabica Coffee via NMR in Relation to the Time and Temperature of the Roasting Procedure

Coffee is one of the most popular and consumed products in the world, with high nutritional value and economic importance. However, some factors can change the organoleptic properties of a coffee species, without causing significant damage such as loss of important components. The present study evaluated the chemical profile, via nuclear magnetic resonance (NMR), of the main biological properties and substances of the drink, verifying similarities in the composition of different types of arabica coffee made in different conditions, such as the roasting time and temperature. The main components were identified, using information from the literature and a database, and compared with the experimental data of 1D and 2D 1H NMR. The spectral data were analyzed and grouped via principal component analysis (PCA) using the Bruker Amix 3.9.14 software. 1H NMR was able to monitor the roasting process and qualify the intact bean and chemical profile of the coffee according to the roasting conditions. Due to the importance of the monitored components, the coffee species analyzed can be identified, along with the appearance of unwanted or adulterating compounds that are normally added to the product to reduce the cost of commercialization.

Understanding the complexation of aliphatic and aromatic acids guests with octa acid

AbstractIn this study, inclusion of 10 guest molecules, adamantyl and naphthyl carboxylic acids with different structural and electronic properties, within a synthetic cavitand octa acid was probed by isothermal calorimetry, 1D and 2D 1H nuclear magnetic resonance spectroscopy, and molecular dynamic simulations. Under the condition of the experiments (pH ~ 8.7), the guests were included as carboxylate anions with the polar anionic head group facing water and hydrophobic carbon skeleton buried within the cavitand, forming 1:1 host to guest complexes. Importance of weak interactions between the guest and the cavitand interior is reflected in the measured negative ΔH values. Although ΔH was negative for all guests, ΔS was positive for adamantyl guests and negative for naphthyl guests. Quite likely the difference in hydrophobicity between the 2 sets of molecules and the strength of interaction between the guest and the host are responsible for the sign difference in ΔS between the 2 series. The importance of steric factor during inclusion of naphthyl carboxylic acids within octa acid cavity is brought out by the difference in thermodynamic parameters between the 1‐ and 2‐substituted naphthyl carboxylic acids; 2‐naphthyl carboxylic acids that can penetrate deeply have larger −ΔH and 1‐naphthyl carboxylic acids that can only enter the cavity in an angle have smaller −ΔH. As expected, based on the well‐known concept of “enthalpy‐entropy compensation,” the molecules that have large −ΔH are accompanied by large −ΔS.

NMR Study Conformations of Calcium Gluconate in the Aqueous Solution

Different spatial structures that arise due to rotation around simple bonds without violating the integrity of the molecule (without breaking chemical bonds) are called conformations. 1H nuclear magnetic resonance (NMR) study of the spatial structure of calcium gluconate in the aqueous solution has been carried out. It was shown that molecules of calcium gluconate exist in the form of two conformations: zigzag 1-P and cyclic 3 G +. The results of homonuclear 2D 1H NMR spectroscopy indicate the predominantly zigzag conformation. It was found that the intermolecular hydrogen bonds are formed and the spatial structure of molecules changes at the increase in the solution concentration. The observed concentration behavior of the conformation of calcium gluconate is also associated with the presence of the intramolecular hydrogen bonds –O(C4)H··O(C2).

Accelerating two-dimensional nuclear magnetic resonance correlation spectroscopy via selective coherence transfer.

Nuclear magnetic resonance (NMR) spectroscopy serves as an important tool for both qualitative and quantitative analyses of various systems in chemistry, biology, and medicine. However, applications of one-dimensional 1H NMR are often restrained by the presence of severe overlap among different resonances. The advent of two-dimensional (2D) 1H NMR constitutes a promising alternative by extending the crowded resonances into a plane and thereby alleviating the spectral congestions. However, the enhanced ability in discriminating resonances is achieved at the cost of extended experimental duration due to necessity of various scans with progressive delays to construct the indirect dimension. Therefore, in this study, we propose a selective coherence transfer (SECOT) method to accelerate acquisitions of 2D correlation spectroscopy by converting chemical shifts into spatial positions within the effective sample length and then performing an echo planar spectroscopic imaging module to record the spatial and spectral information, which generates 2D correlation spectrum after 2D Fourier transformation. The feasibility and effectiveness of SECOT have been verified by a set of experiments under both homogeneous and inhomogeneous magnetic fields. Moreover, evaluations of SECOT for quantitative analyses are carried out on samples with a series of different concentrations. Based on these experimental results, the SECOT may open important perspectives for fast, accurate, and stable investigations of various chemical systems both qualitatively and quantitatively.

Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) 1H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D 1H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.

Effects of time and temperature of firing on Fe-rich ceramics studied by Mössbauer spectroscopy and two-dimensional 1H-nuclear magnetic resonance relaxometry

The combined effects of firing temperature and soaking time on the microstructure of iron-rich porous ceramics have been studied by 57Fe-Mössbauer spectroscopy and 2D 1H nuclear magnetic resonance (NMR) relaxometry using a single-sided probe. Examining water-saturated ceramics using the relaxation correlation method, where longitudinal (T1) and transverse (T2) relaxation times are measured concurrently, provides information about firing-induced changes in both porosity (related to T1) and magnetic properties (related to T2). Comparing the information obtained from 1H-NMR analyses with that obtained from Mössbauer spectroscopy (which characterizes changes in iron-bearing species) shows that the T1-T2 NMR correlation technique is very sensitive to even subtle modifications in the magnetic behavior of Fe-bearing species. Moreover, the single-sided NMR approach allows us to perform millimeter-scale depth-resolved measurements, which can be used to non-invasively study the microstructural heterogeneities associated with non-uniform firing effects inside ceramics. This is in contrast to Mössbauer spectroscopy, which requires that the ceramic samples be ground.

Heparin sodium compliance to USP monograph: Structural elucidation of an atypical 2.18ppm NMR signal

The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years.

HIV-1 p6—Another viral interaction partner to the host cellular protein cyclophilin A

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D 1H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl–prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme–substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

Structural elucidation of an -1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.

Use of 2-hydroxypropyl-β-cyclodextrin as adjuvant for enhancing encapsulation and release characteristics of asiaticoside within and from cellulose acetate films

The inclusion complex between asiaticoside (AC), an active substance from the medicinal plant Centella asiatica L., and 2-hydroxypropyl-β-cyclodextrin (HPβCD) was investigated. The phase solubility profiles of AC in distilled water and 90:10 (v/v) mixture of 80 vol.% acetic acid and N,N-dimethylacetamide (DMAc) both in the absence and the presence of HPβCD were classified as A L-type and indicated a 1:1 molar ratio of the AC/HPβCD complex. The 1:1 stoichiometric molar ratio of the complex was confirmed by both the 1D and the 2D 1H-nuclear magnetic resonance spectrometry. Unlike the highly crystalline nature of the native AC, the stoichiometric AC/HPβCD complex powder was amorphous in nature. The average diameters of the self-assembling aggregates of the AC/HPβCD complex, a priori prepared at various HPβCD to AC molar ratios of 0.5, 1, and 2, in the solution state, were about 2.4, 3.3, and 3.8 μm, respectively. The solvent-cast CA films containing these complexes showed strong evidence of the aggregates on their surfaces. The maximal cumulative released amounts of AC, a sparingly water-soluble substance, from the CA films containing a mixture of AC/HPβCD of varying molar ratios (i.e., 1:0.5, 1:1, and 1:2) in the phosphate buffer saline solution (PBS) containing 10% (v/v) methanol (i.e., P/B/M medium) were 2.2, 3.9, and 5.8% (based on the actual weights of the film specimens), respectively, which were clearly an increasing function of the HPβCD content in the films.

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation.

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.

Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions.

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.

Retention of the Cis Proline Conformation in Tripeptide Fragments of Bovine Pancreatic Ribonuclease A Containing a Non-natural Proline Analogue, 5,5-Dimethylproline

Attention is focused on l-5,5-dimethylproline (dmP) as a substitute to lock l-proline (Pro) in a cis conformation in peptides and proteins, to prevent cis/trans isomerization when a protein with cis X-Pro peptide groups unfolds. Procedures have been developed to obtain optically pure l-dmP and to incorporate this sterically hindered residue as the central one in tripeptides that are suitable for fragment coupling to prepare synthetic proteins. Based on the sequences of residues 92−94 (Tyr-Pro-Asn:YPN) and 113−115 (Asn-Pro-Tyr: NPY) in bovine pancreatic ribonuclease A (RNase A), in which the X-Pro peptide groups are in the cis conformation, the tripeptides Ac-Tyr-dmP-Asn (YdmPN) and Ac-Asn-dmP-Tyr (NdmPY) were synthesized, and their structures were determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. YdmPN was found to exist solely in the cis conformation between 6 and 60 °C, whereas NdmPY was found to have some trans component that increased from about 10% to about 21% as the temperature increased over the range between 6 and 80 °C. Both YdmPN and cis-NdmPY adopt a type VI reverse turn, as does proline. The NMR structures of YdmPN and cis-NdmPY are comparable with the X-ray structures of the corresponding portions of RNase A, and the NMR structure of trans-NdmPY is compatible with the X-ray structure of the isolated tripeptide, Ac-NPY. These results demonstrate that l-dmP is a promising substitute for proline in a variety of protein problems to constrain the X-Pro peptide group to the cis conformation.

1H NMR structure of an antifungal γ-thionin protein SIα1: Similarity to scorpion toxins

The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.

1H NMR spectroscopic imaging of myocardial triglycerides in excised dog hearts subjected to 24 hours of coronary occlusion.

Myocardial ischemic insult causes depression of fatty-acid beta-oxidation and increased fatty-acid esterification with triglyceride (TG) accumulation. This accumulation has been demonstrated to occur in the territory with diminished blood flow surrounding an infarct, ie, the region at risk. To evaluate whether the extent of TG accumulation in the canine heart after 24 hours of ischemia could be detected, we applied myocardial 1H nuclear magnetic resonance (NMR) spectroscopic imaging (SI). Seven adult mongrel dogs underwent 24 hours of left anterior descending coronary artery occlusion. Postmortem, the hearts were excised and the size and location of the infarct were determined. With a Philips 1.5-T clinical NMR imaging/spectroscopic system, two-dimensional (2D) 1H NMR SI was performed. TG 1H NMR chemical shift images were reconstructed from the frequency domain spectra by numerical integration. A statistically significant (P < .05) increase in TG signal intensity was demonstrated in the region at risk compared with the nonischemic control region. There was an intermediate quantity of TG in the infarct region. Biochemical determination of tissue TG content (milligrams per gram wet weight) in the control, at-risk, and infarct regions confirmed the 1H NMR measurements. Histological evaluation with oil red O staining also demonstrated graded TG accumulation in myocytes. The highest TG levels were found in the at-risk region and the lowest levels in the control region. By use of 2D 1H NMR SI, the present study confirms and extends previous work that demonstrates preferential accumulation of TG in the reversibly injured myocardium after 24 hours of coronary occlusion. This study provides an important step toward the clinical application of TG imaging. When TG imaging is ultimately possible, resultant data would have diagnostic, prognostic, and therapeutic implications.

Structural identification of paramagnetic iron porphyrins from Colorado coal with the use of 1D and 2D 1H NMR spectroscopy: geochemical implications on their origin from heme

A fraction of iron porphyrins from Colorado coal constituting one major and one minor C28 isomer was analysed with the use of 1D and 2D 1H nuclear magnetic resonance spectroscopy. 2D 1H COSY and NOESY experiments in a low viscosity solvent (CD 3 OD) led to the structrural identification of the major component. Assignment of the hyperfine shifted resonances, of all the peripheral substituents on the porphyrin macrocycle, resulted in an unambiguous identification of iron(III) 2,7,12,18-tetramethyl-13,17-diethyl porphyrin. Reliability of the 2D 1H NMR method used was confirmed by analyzing a related structure, i.e. deuteroheme-IX in the same way. The minor constituent of the analysed fraction was identified by reproducing the 1H NMR spectrum with its synthetic counterpart: low-spin iron(III) 2,7,12,18-tetramethyl-3,8-diethyl porphyrin. Both identified coal iron porphyrins represent products derived from a common protoheme-IX related precursor, selectively degraded via different routes during coal diagenesis.

Secondary structure of fibronectin type 1 and epidermal growth factor modules from tissue-type plasminogen activator by nuclear magnetic resonance.

A segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution. The sequence-specific assignment of the 1H and 15N nuclear magnetic resonances from the pair of modules has been completed using 2D 1H nuclear magnetic resonance (NMR) spectra in conjunction with 3D, 15N-edited, 1H and 2D 15N-1H NMR spectra. Slowly exchanging amide protons have been identified, and estimates of a number of backbone 3JNH-C alpha H coupling constants were obtained by line-shape-fitting. The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the "consensus" G module structure determined previously from several isolated G modules. In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module. The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module. The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22.

Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy.

The solution structures of the recently discovered neuropeptides PACAP38 and PACAP27 have been investigated in aqueous solution containing varying amounts of trifluoroethanol (TFE) by circular dichroism (CD) spectroscopy and a combination of 2D 1H nuclear magnetic resonance (NMR) spectroscopy, distance geometry, and refined molecular dynamics and energy minimization calculations. In aqueous solution both peptides show only small transitory amounts of stable structure while in 50% TFE they adopt ordered structures. Qualitative NOE data and the use of the chemical shift index of the alpha-protons identified the positions of alpha-helical regions. A set of low-energy conformations compatible with the quantitative NOE data were obtained for both and each set were subjected to RMS analysis to determine the positions of the secondary structure elements. PACAP38 has an initial disordered N-terminal domain of eight amino acids, followed by an alpha-helical structure stretching from Ser-9 to Val-26, which contains a discontinuity between Lys-20 and Lys-21, and in the C-terminal region there is a short alpha-helix between Gly-28 and Arg-34. The structure of PACAP27 mirrors remarkably closely that of PACAP38 and shows no fraying of the C-terminal helix. The physiological significance of the three structural domains (1-8, 9-26, and 27-38) of PACAP38 is shown by a comprehensive review of recent in vitro and in vivo investigations of PACAP analogues. The correspondence of the global structural features of PACAP with other members of this family of peptides (namely, secretin, glucagon, GHRF1-29 and VIP) is demonstrated by inspection of the chemical shift indices of the alpha-protons.

A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding.

The effect of 2,2,2-trifluoroethanol (TFE) on the solution conformation of hen egg white lysozyme has been investigated using circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. Addition of TFE to lysozyme at pH 2.0, 27 degrees C, up to a concentration of 15% (v/v) induces only slight changes in the NMR spectrum. However, above this concentration a cooperative transition to a new but partially structured state of the protein is observed. This state shows no structural cooperativity against further denaturation and is characterized by an ellipticity in the far-UV CD greater than that of the native protein. Near-UV CD intensity is dramatically reduced compared with that of the native state, and 1H NMR studies indicate that side-chain interactions are substantially averaged in this denatured state. Solvent proton/deuterium exchange rates for 66 amide hydrogens were measured site-specifically by a combination of amide trapping experiments and 2D 1H NMR. Significant protection from exchange occurs for about 25 backbone amides, the majority of which are located in regions of the protein that are helical in the native enzyme. By contrast, amides located in a second region of the native protein which contains a beta-sheet and one 3(10)-helix as well as a long loop show little protection. This pattern of protection resembles that found in the stable molten globule state of alpha-lactalbumin and in an early kinetic intermediate detected in the refolding of hen lysozyme.

1H nuclear magnetic resonance investigation of cobalt(II) substituted carbonic anhydrase

The structure of ClO4 and NO3 adducts of cobalt(II) substituted bovine carbonic anhydrase have been investigated through 1D NOE and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. For the first time two-dimensional NMR techniques are applied to paramagnetic metalloproteins other than iron-containing proteins. Several active site signals have been assigned to specific protons on the grounds of their scalar and dipolar connectivities and T1 values. The experimental dipolar shifts for the protons belonging to noncoordinated residues have allowed the identification of a plausible orientation of the magnetic susceptibility tensor around the cobalt ion as well as of the magnitude and the anisotropy of the principal susceptibility values. In turn, a few more signals have been tentatively assigned on the grounds of their predicted dipolar shifts. The two inhibitor derivatives have a very similar orientation but a different magnitude of the chi tensor, and the protein structure around the active site is highly maintained. The results encourage a more extensive use of the two-dimensional techniques for obtaining selective structural information on the active site of metalloenzymes. With this information at hand, comparisons within homologous series of adducts with various inhibitors and/or mutants of the same enzyme of known structure should be possible using limited sets of NMR data.