Long range hydrogen bond mediated effects in peptides: nitrogen-15 NMR study of gramicidin S in water and organic solvents

Live, David; Davis, Donald G.; Agosta, William C.; Cowburn, David

doi:10.1021/ja00319a006

Cited by 227 publications

(148 citation statements)

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“…A polynomial baseline correction was applied in the directly detected dimension. The 1 H frequency scale of all spectra was directly referenced to d 4 -TSP at 0.00 ppm, while the 15 N frequency scale was indirectly referenced to liquid NH 3 by using the method described by Live et al (39).…”

Section: Methodsmentioning

confidence: 99%

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

Matthews

Young

Tucker³

et al. 2000

J Virol

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Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K 3 Ϸ 2.2 ؋ 10 11 M ؊2 ) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with ␤-sheet-like characteristics. Only a small increase in ␣-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999). Finally, HR3 forms weak ␣-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.

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Section: Methodsmentioning

confidence: 99%

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

Matthews

Young

Tucker³

et al. 2000

J Virol

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“…Alternatively, refocusing delays were incorporated to eliminate the need for any phase correction. Nitrogen and carbon chemical shifts were referenced with respect to external standards as described previously (Live et al, 1984) using liquid ammonia and tetramethylsilane as chemical shift standards. Additional details of the data collection for all NMR experiments are summarized in Table 2.…”

Section: Nmr Methods: Experimentsmentioning

confidence: 99%

Heteronuclear (¹H, ¹³C, ¹⁵N) NMR assignments and secondary structure of the basic region‐helix‐loop‐helix domain of E47

1997

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E47 is an immunoglobulin enhancer DNA-binding protein that contains a basic region-helix-loop-helix (blHLH) domain. This structural motif defines a class of transcription factors that are central to the developmental regulation of many tissues. Its function is to provide a dimerization interface through the formation of a parallel four-helix bundle, resulting in the juxtaposition of two basic DNA-recognition a-helices that control sequence-specific DNA-binding. In order to gain insight into the biophysical nature of b/HLH domains, we have initiated structural studies of the E47 homodimer by NMR. Sequence-specific resonance assignments have been obtained using a combination of heteronuclear double-and triple-resonance NMR experiments. The secondary structure was deduced from characteristic patterns of NOEs, '3Calp chemical shifts, and measurements of 3JHNHa scalar couplings. Except for the basic region recognition helix, the secondary structural elements of the E47 homodimer are preserved in the absence DNA when compared with the co-crystal structure of E47 bound to DNA (Ellenberger T, Fass D, Arnaud M, Harrison SC, 1994, Genes & Dev 8:970-980). As expected, the DNA-binding helix is largely unstructured, but does show evidence of nascent helix formation. The HLH region of E47 is structured, but highly dynamic as judged by the rapid exchange of backbone hydrogen atoms and the relatively weak intensities of many of the NOEs defining the dimerization helices. This dynamic nature may be relevant to the ability of E47 both to homodimerize and to heterodimerize with MyoD, Id, and Tall. Abbreviations: PMSF, phenyl methyl sulfonyl fluoride; DTT, dithiothreitol; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single-quantum coherence; CT, constant time; ID, 2D, 3D, 4D, one-, two-, three-, and four-dimensional NMR; P P I , time-proportional phase incrementation; Tip, rotating frame relaxation time; T i , the arithmetic mean of zero quantum and double quantum relaxation times; 'H-'D exchange, hydrogen deuterium exchange. tion domain capable of forming dimers, tetramers, and higherorder aggregates. Dimerization results in the pairing up of each HLH motif to form a four-helix bundle. Upon dimerization, this domain brings together two N-terminal arginine-and lysine-rich DNA-binding a-helices (the basic region), which promotes specific binding to the pseudo-symmetric CANNTG DNA sites found in the E-box control regions of many developmentally regulated genes.

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“…6) shows a single set of crosspeaks corresponding to the monomer primary structure, indicating that the peptide 15N-and relevant lH-NMR chemical shifts for homodimeric rJunLZ in H,O solution at pH3.6 and 310K. The 'Hfrequency scale was directly referenced to 3-trimethylsilyl(2,2,3,3-2H,)propionate at 0.00 ppm, while the "N-frequency scale was indirectly referenced to liquid NH, by using the ' H frequency of the above standard resonance (Live et al, 1984 This conclusion is reinforced by the observation of only a single crosspeak for the backbone amide group of each residue in the 15N-'H HSQC spectrum of uniformly '5N-labelled rJunLZ (see Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

“…A total of 512 tl increments of 4096 complex data points were collected using 32 transients per t, increment. The 'H-frequency scale was referenced directly to 3-trimethylsilyl(2,2,3,3-'HJpropionate at 0.00 ppm, while the "N-frequency scale was indirectly referenced to liquid NH, by using the 'H frequency of the 3-trimethy1si1y1(2,2,3,3-*H4)propionate resonance (Live et al, 1984). 2D spectra were processed using UXNMR software running on either a Bruker Aspect X32 or a Silicon Graphics Indigo R4000 workstation.…”

Section: Nmr Experimentsmentioning

confidence: 99%

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain

et al. 1994

European Journal of Biochemistry

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Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of cJun plus a total of five engineered non-native residues at the N-and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (~1 5 % total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carbuxy-terminal portion of Schistosorna japonicum glutathione Stransferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel a-helices. We also present I5N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.Jun and Fos are members of a family of eukaryotic transcriptional activators that contain a leucine zipper motif, which is characterized by a heptad repeat of leucine residues (Landschulz et al., 1988). It was originally postulated that the Jun and Fos leucine zipper segments form extended ahelices and that the highly hydrophobic leucine side chains projecting from a Jun helix interdigitate with those of an antiparallel Fos helix to form a heterodimer, thus creating an analogy with the interlocking of teeth on a zipper. This leucine zipper motif was proposed to juxtapose the adjacent basic regions of the two polypeptides in a manner suitable for recognition of a specific palindromic DNA recognition sequence (5'-TGACTCA-3'; Neuberg et al., 1989) known as the transcriptional regulatory element or AP-1 binding site. More recent studies of these proteins indicate that the helical leucine zipper domains associate in parallel (Gentz et al., 1989) and that the zipper itself is probably a short coiled-

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Long range hydrogen bond mediated effects in peptides: nitrogen-15 NMR study of gramicidin S in water and organic solvents

Cited by 227 publications

References 0 publications

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

Heteronuclear (¹H, ¹³C, ¹⁵N) NMR assignments and secondary structure of the basic region‐helix‐loop‐helix domain of E47

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain

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Long range hydrogen bond mediated effects in peptides: nitrogen-15 NMR study of gramicidin S in water and organic solvents

Cited by 227 publications

References 0 publications

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

Heteronuclear (1H, 13C, 15N) NMR assignments and secondary structure of the basic region‐helix‐loop‐helix domain of E47

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain

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Heteronuclear (¹H, ¹³C, ¹⁵N) NMR assignments and secondary structure of the basic region‐helix‐loop‐helix domain of E47