Stable isotope-labeled vitamin D, metabolites and chemical analogs: synthesis and use in mass spectrometric studies

Methods for the measurement of vitamin D and its metabolites using stable isotope-labeled internal standards and mass spectrometry are reviewed. The synthesis of both labeled and unlabeled standards is illustrated, and details of the synthesis of (26,26,27,27,27(-2)H5)-25,26-dihydroxyvitamin D3 and (28,28,28(-2)H3)-24,25-dihydroxyvitamin D2 are given. The use of in vitro biologic systems for the production of further metabolites of deuterated 25-hydroxyvitamin D3 is discussed. Use of deuterated 25-hydroxydihydrotachysterol3 as a substrate in the isolated perfused rat kidney has provided valuable data for the assignment of structure to a number of metabolites of 25-hydroxydihydrotachysterol3 formed in this system.     

Backbone assignment of proteins with known structure using residual dipolar couplings

A prerequisite for NMR studies of protein-ligand interactions or protein dynamics is the assignment of backbone resonances. Here we demonstrate that protein assignment can significantly be enhanced when experimental dipolar couplings (RDCs) are matched to values back-calculated from a known three-dimensional structure. In case of small proteins, the program MARS allows assignment of more than 90% of backbone resonances without the need for sequential connectivity information. For bigger proteins, we show that the combination of sequential connectivity information with RDC-matching enables more residues to be assigned reliably and backbone assignment to be more robust against missing data. Structural or dynamic deviations from the employed 3D coordinates do not lead to an increased error rate in RDC-supported assignment. RDC-enhanced assignment is particularly useful when chemical shifts and sequential connectivity only provide a few reliable assignments.     

GFT NMR Experiments for Polypeptide Backbone and 13Cβ Chemical Shift Assignment

(4,3)D, (5,3)D and (5,2)D GFT triple resonance NMR experiments are presented for polypeptide backbone and (13)C(beta) resonance assignment of (15)N/(13)C labeled proteins. The joint sampling of m = 2, 3 or 4 indirect chemical shift evolution periods of 4D and 5D NMR experiments yields the measurement of 2(m) - 1 linear combinations of shifts. To obtain sequential assignments, these are matched in corresponding experiments delineating either intra or interresidue correlations. Hence, an increased set of matches is registered when compared to conventional approaches, and the 4D or 5D information allows one to efficiently break chemical shift degeneracy. Moreover, comparison of single-quantum chemical shifts obtained after a least squares fit using either the intra or the interresidue data demonstrates that GFT NMR warrants highly accurate shift measurements. The new features of GFT NMR based resonance assignment strategies promise to be of particular value for establishing automated protocols.     

Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a

The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1H and 15N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.     

1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.     

Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial ¹⁵N‐, ¹³C^α‐, and ¹³C'‐selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell‐free expression system, a scheme that involves ¹⁵N, 1‐¹³C, 2‐¹³C, fully ¹⁵N/¹³C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time‐shared triple‐resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non‐proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide ¹H and ¹⁵N resonances need to be obtained, even in cases where sensitivity is the limiting factor.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.     

2D NMR studies of aminoglycoside antibiotics. Use of relayed coherence transfer for 1H resonance assignment and in situ structure elucidation of amikacin derivatives in reaction mixtures

Phase-sensitive 2D 1H/1H COSY spectra can be used to identify the structures of individual pure specimens of the aminoglycoside antibiotic amikacin and its N-hemisuccinyl derivatives. However, even at 500 MHz the 2D chemical shift dispersion does not allow for unambiguous assignment of all cross-peaks. By use of 2D relayed coherence transfer experiments (RELAY) optimized to detect two-step 1H/1H scalar interactions in which one of the J-values is small, sufficient additional correlations can be obtained from the frequency-isolated resonances to allow facile tracing of all scalar connectivities. Complete assignments of the 1H NMR spectra of amikacin, its 6'-N-hemisuccinamide, and a novel bis(acylate) [gamma-N-(p-vinylbenzoyl)amikacin 6'-N-hemisuccinamide] were obtained for aqueous media. The NMR spectrum of amikacin free base was also assigned in dimethyl sulfoxide solution. The RELAY experiment can be extended to the analysis of reaction mixtures, which allows for the identification and resonance assignment of regioisomeric amikacin haptens in the mixture state. All of the N-monohemisuccinyl isomers of amikacin have been identified in reaction mixtures through the RELAY experiment. The relative reactivities of the amino functions of amikacin toward acylating agents were found to be 6'-N greater than 3-N equal to or greater than 3"-N equal to or greater than gamma-N. However, this reactivity order is altered after the initial acylation event.     

Tritium isotope effect in high-pressure liquid chromatography of vitamin D metabolites

A significant chromatographic isotope effect is reported for 1,25-dihydroxyvitamin D3 in a wide variety of HPLC separation systems. The effect is also observed for 24,25-dihydroxyvitamin D3. Retention times differ from less than 1% up to 4% depending on the separation system and the degree and position of tritium substitution. Such an effect must be corrected for whenever both labeled and unlabeled vitamin D metabolites are used in HPLC cochromatography or assay recovery studies.     

13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).     

Assignment of the gene responsible for cystinuria (rBAT) and of markers D2S119 and D2S177 to 2p16 by fluorescence in situ hybridization

We have established rBAT (named as SLC3A1 in the Genome Data Base) as a gene responsible for cystinuria, a heritable disorder of amino acid transport. The cystinuria locus has been mapped by linkage between microsatellite markers D2S119 and D2S177. Fluorescene in situ hybridization (FISH) either with Alu-polymerasechain-reaction (PCR)-amplified sequences of a yeast artificial chromosome (YAC) containing the rBAT gene or with rBAT-specific PCR-amplified genomic fragments, and chromosome G-banding have cytogenetically mapped rBAT to 2p16.3. In order to correlate the physical and genetic information on cystinuria, we have performed FISH with combinations of Alu-PCR- amplified sequences from YACs containing rBAT or the D2S119 and D2S177 loci. In all cases, a fused signal is obtained that demonstrates their close physical location; this allows the assignment of rBAT, cystinuria and their linked markers, D2S119 and D2S177, to 2p16.     

Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Towards automatic protein backbone assignment using proton-detected 4D solid-state NMR data

We introduce an efficient approach for sequential protein backbone assignment based on two complementary proton-detected 4D solid-state NMR experiments that correlate \( {\text{H}}_{{\text{i}}}^{{\text{N}}} \) /N_i with CA_i/CO_i or CA_i?1/CO_i?1. The resulting 4D spectra exhibit excellent sensitivity and resolution and are amenable to (semi-)automatic assignment approaches. This strategy allows to obtain sequential connections with high confidence as problems related to peak overlap and multiple assignment possibilities are avoided. Non-uniform sampling schemes were implemented to allow for the acquisition of 4D spectra within a few days. Rather moderate hardware requirements enable the successful demonstration of the method on deuterated type III secretion needles using a 600 MHz spectrometer at a spinning rate of 25 kHz.     

4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on ¹H^β and ¹³C^δ efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13 kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCH^aro sequence are proposed.     

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and ¹H and ¹³C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding ¹³C resonances, which provides an important step toward automated structure determination of RNAs.     

SCAssign: a sparky extension for the NMR resonance assignment of aliphatic side-chains of uniformly 13C,15N-labeled large proteins

SCAssign (side-chain assignment) is a Sparky extension written in Python to assist the NMR resonance assignment of aliphatic side-chains of uniformly (13)C,(15)N-labeled large proteins. It is based on a general strategy recently developed in our laboratory that makes use of 4D (13)C,(15)N-edited NOESY, 3D MQ-(H)CC(m)H(m)-TOCSY, and prior backbone assignments. The program runs on all operating systems for which Sparky is available, and is easy to install, setup and use. Not only can it accelerate the assignment process, it also allows assignments of weak NOEs in 4D NOESY, which used to be very difficult with manual approach. AVAILABILITY: The program, in the form of source code, is provided as free download at http://yangdw.science.nus.edu.sg/SCAssign. The website also contains installation guide, user manual and demonstrations recorded in Flash.     

Protein fold recognition using sequence-derived predictions.

In protein fold recognition, one assigns a probe amino acid sequence of unknown structure to one of a library of target 3D structures. Correct assignment depends on effective scoring of the probe sequence for its compatibility with each of the target structures. Here we show that, in addition to the amino acid sequence of the probe, sequence-derived properties of the probe sequence (such as the predicted secondary structure) are useful in fold assignment. The additional measure of compatibility between probe and target is the level of agreement between the predicted secondary structure of the probe and the known secondary structure of the target fold. That is, we recommend a sequence-structure compatibility function that combines previously developed compatibility functions (such as the 3D-1D scores of Bowie et al. [1991] or sequence-sequence replacement tables) with the predicted secondary structure of the probe sequence. The effect on fold assignment of adding predicted secondary structure is evaluated here by using a benchmark set of proteins (Fischer et al., 1996a). The 3D structures of the probe sequences of the benchmark are actually known, but are ignored by our method. The results show that the inclusion of the predicted secondary structure improves fold assignment by about 25%. The results also show that, if the true secondary structure of the probe were known, correct fold assignment would increase by an additional 8-32%. We conclude that incorporating sequence-derived predictions significantly improves assignment of sequences to known 3D folds. Finally, we apply the new method to assign folds to sequences in the SWISSPROT database; six fold assignments are given that are not detectable by standard sequence-sequence comparison methods; for two of these, the fold is known from X-ray crystallography and the fold assignment is correct.     

Domain 3 of kininogens contains a cell-binding site and a site that modifies thrombin activation of platelets.

High and low molecular weight kininogens (HK and LK) are able to bind to platelets to inhibit thrombin binding to and activation of platelets. The heavy chain domain on the kininogens that contains these functions has been determined. Domain 3 (D3) but not domains 1 or 2, completely inhibited 125I-HK binding to platelets (Ki = 24 +/- 7 nM, n = 4). 125I-D3 specifically bound to unstimulated platelets and human umbilical vein endothelial cells. On platelets, it was blocked by unlabeled D3 and HK but not prekallikrein, factor XII, C1s, or C1 inhibitor. Further, one monoclonal antibody (HKH13) directed to kininogens' D3 blocked 125I-HK and 125I-D3 binding to platelets. The binding of 125I-D3 to platelets was fully reversible by addition of 35 molar excess of unlabeled D3. D3 binding to platelets was saturable with an apparent Kd of 39 +/- 8 nM (n = 4) and 1227 +/- 404 binding sites/platelet. D3, like HK and LK, inhibited thrombin-induced platelet activation by preventing thrombin binding to platelets. Another monoclonal antibody (HKH12), directed to D3, which did not block HK binding to platelets, reduced HK's ability to inhibit 125I-alpha-thrombin binding. This result suggests that the region on D3 that inhibits 125I-alpha-thrombin binding to platelets is different from that which directly binds to platelets. These studies indicate that D3 of the kininogens contains both a binding region for platelets and endothelial cells and another region that inhibits thrombin-induced platelet activation.