HNCA Research Articles

Secondary structure and topology of interleukin-1 receptor antagonist protein determined by heteronuclear three-dimensional NMR spectroscopy.

Interleukin-1 (IL-1) proteins, such as IL-1 beta, play a key role in immune and inflammatory responses. Interaction of these cytokines with the IL-1 receptor induces a variety of biological changes in neurologic, metabolic, hematologic, and endocrinologic systems. Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. The 153-residue protein binds to the receptor with an affinity similar to that of IL-1 beta but does not elicit any physiological responses. As a first step toward understanding IRAP's mode of action, we have used multidimensional, heteronuclear NMR spectroscopy to determine the antagonist's solution secondary structure and global fold. Using a combination of 3D 1H-15N NOESY-HMQC and TOCSY-HMQC and 3D 1H-15N-13C HNCA and HN(CO)CA experiments on uniformly 15N- or doubly 13C/15N-enriched IRAP, we have made resonance assignments for more than 90% of the main-chain atoms. Analysis of short- and long-range NOE's indicates that IRAP is predominantly beta-sheet, with the same overall topology as IL-1 beta but with different regions of the primary sequence comprising the beta-strands. Two short helical segments also were identified. The 14% sequence identity between IL-1 beta and IRAP increases to 25% when differences in the locations of secondary structure elements in the primary sequences are taken into account. Still, numerous differences in side chains, which ultimately play a major role in receptor interaction, exist.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR

A new three-dimensional triple resonance NMR experiment is described that correlates the amide 1 H and 15 N resonances of one residue simultaneously with both the 13 C α and 13 C β resonances of its preceding residue. Sensitivity of the new experiment is comparable with that of the HN(CO)CA experiment (Bax, A.; Ikura, M.J. Biomol. NMR 1991, 1, 99-105), but the additional correlation to the C β resonance of the preceding residue provides invaluable assignment information, previously inaccessible

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1H N, 15N, and 13C′ chemical shifts in 15N 13C-labelled proteins

Sequence-specif ic resonance assignments are a prerequisite for structural and dynamical interpretation of protein NMR spectra. For proteins smaller than 10 kDa assignment strategies have relied upon through-bond correlations in homonuclear COSY and TOCSY spectra to identify resonances associated with particular spin systems. Conformation-dependent nuclear Overhauser effects are then emp loyed to sequentially connect these spin systems (1-5). In larger proteins, however, extensive resonance overlap and decreased sensitivity of experiments utilizing ‘H‘H scalar couplings have hindered this approach. ‘HI%15N triple-resonance experiments provide a conformation-independent approach for the assignment of backbone resonances in I%15N-labeled large proteins (6-13). In addition these experiments allow accurate measurement of coupling constants in proteins with large linewidths (14, 15). These experiments exploit large heteronuclear one-bond couplings to transfer magnetization with the sensitivity of indirect detection. As demonstrated in calmodulin ( 16, 17) backbone assignments utilize four tr iple-resonance experiments [ HNCa, HNCO, HCaCO, and HCA(CO)N] and the 3D TOCSY-HMQC experiment ( 18). A fifth tr iple-resonance experiment [ H( CA)NHN] is a useful complement to the TOCSY-HMQC experiment, correlating ‘HN “N and ‘H” resonances ( 7, 9, 13). Furthermore, a sixth experiment, the HN (CO ) CA. has been introduced, providing additional sequential information ( 11) . Of the experiments listed above, those which detect am ide protons [ HNCA, HNCO, H(CA)NHN, HN(CO)Ca] need to be collected in H20, while those with detection of (Y protons [ HCACO and HCA( CO)N] are best performed in 2H20. This causes difficulties if assignments are based on al ignment of C’ chemical shifts, because these resonances may show significant isotope shifts, depending on whether the carbonyl is hydrogen bonded to a proton or a deuteron. It is therefore desirable to perform the ma jority of experiments in the same solvent ( H20), restricting oneself to those experiments which detect am ide protons [ HNCA, HNCO, HN( CO)CA. H( CA )NHN 1.

A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

A 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C, 15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.

Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein

Recently proposed 3D triple-resonance techniques (I-5) make it possible to obtain sequential assignment of the backbone ‘H, 15N and 13C resonances in proteins that can be isotopically labeled with 13C and “N. Thi’s approach has been used successfully for a number of systems, including calmodulin ( 16.7 kDa) ( 1) , calmodulin complexed with a 26-residue peptide ( -20 kDa) (6), and the phospho-carrier protein IIIG’” ( 7) ( 18 kDa). All of these triple-resonance experiments rely on transfer of magnetization via heteronuclear one-bond Jcouplings and the sensitivity of the experiments depends strongly on the ratio of the size of the J coupling and the linewidths of the nuclei involved in the magnetization transfer process. Our original experiments were designed to minimize the number of RF pulses required for a particular pulse sequence, and thus to minimize the effect of pulse imperfections. Subsequent experience with these experiments has indicated that the effect of pulse imperfections is not as severe as originally expected, provided that pulses are properly calibrated. Hence, in order to optimize the experiments it is more important to minimize the relaxation of transverse magnetization by keeping the dephasing and rephasing delays as short as possible, especially for larger proteins. Here we demonstrate the applicability of three improved triple-resonance experiments to the study of interferon-y, a dimer with 134 residues per monomer and a total molecular weight of 3 1.4 kDa. Correlation of the amide proton and “N resonance with the intraresidue 13Ca can be obtained with an experiment known as HNCA (I, 2). Note that this experiment also provides connectivity to the Ca of the preceding residue, transferring coherence via the ’ JN-Ca coupling. Because ’ JN-Ca and ’ JNxn can be of comparable magnitude (8) it may not always be possible to distinguish intrafrom interresidue connectivities with this experiment. In contrast, the HN( CO)CA experiment (4) provides correlations exclusively to the Ca resonance of the preceding residue by relaying magnetization via the intervening carbonyl 13C spin. The HN( CO)CA experiment is closely related to the HNCO experiment, which correlates the amide ‘H and “N with the 13C of the preceding residue. In the present study of interferon-y, we employ modified versions of these experiments, using INEPT (9) instead of HMQC (10-12) type coherence transfers, and overlay the 13C15N dephasing period with a 15N evolution period

An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15N/13C enriched proteins.

A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C alpha chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from 15N to 13C alpha via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.

The design and optimization of complex NMR experiments. Application to a triple-resonance pulse scheme correlating Hα, NH, and 15N chemical shifts in 15N 13C-labeled proteins

Sequence-specific assignments of the backbone resonances of proteins form the basis for further study of the structural and dynamic properties of the molecule under investigation. Traditional assignment strategies have relied on through-bond and through-space connectivities provided by homonuclear COSY, HOHAHA/TOCSY, and NOESY spectra (I-4). While very fruitful for proteins less than 10 kDA, this approach becomes difficult for larger systems, due to extensive overlap and decreasing sensitivity of experiments relying on through-bond connectivities. Recently, we have proposed a novel approach for the sequential assignment of 'H, 13C, and 15N spectra of larger proteins based on triple-resonance three-dimensional NMR spectroscopy (5, 6). These experiments exploit the relatively large one-bond J couplings between the backbone 13C and “N nuclei and between the backbone protons and the 13C and “N nuclei to which they are directly attached. Because the couplings are often large compared to pertinent linewidths for proteins less than 25 kDa in molecular weight, magnetization can be transferred between spins in an efficient manner, with the resultant spectra having good sensitivity. Moreover, because the experiments are recorded in the “3D mode,” spectral overlap is virtually eliminated. The sequential assignment of 13C-15N-labeled proteins using the triple-resonance approach is aided considerably by the HOHAHA-HMQC experiment (7). For example, the HNCA triple-resonance experiment correlates the NH and r5N chemical shifts with the intraresidue Ccu chemical shift. When combined with the HNCA experiment, the HOHAHA-HMQC experiment (which correlates “N, NH, and Ha chemical shifts) firmly establishes intraresidue correlations between pairs of “N-NH and G-Ha backbone resonances, despite significant overlap in 1 D ‘H, 15N, and 13C spectra. Linking the 15N, NH, CCX, and Ha chemical shifts is the first step in the sequential assignment process using this new approach. Unlike the triple-resonance experiments, the HOHAHA-HMQC experiment is very sensitive to the secondary structure of the protein under study, as the efficiency of magnetization transfer between NH and Ha protons depends strongly on the NH-Ha scalar couplings. These couplings are, in turn, strongly related to the backbone angle I#I and are less than 6 Hz in regions of regular a-helical secondary structure. For larger proteins rich in a-helical content, a substantial number of NH-Ha connectivities are often found to be weak or missing. In this paper a triple-