Nuclear magnetic resonance studies on yeast tRNAPhe. II. Assignment of the iminoproton resonances of the anticodon and T stem by means of nuclear Overhauser effect experiments at 500 MHz.

Resonances of the water exchangeable iminoprotons of the T and anticodon stem of yeast tRNAPhe were assigned by means of Nuclear Overhauser Effects (NOE's). Together with our previous assignments of iminoproton resonances from the acceptor and D stem (A. Heerschap, C.A.G. Haasnoot and C.W. Hilbers (1982) Nucleic Acids Res. 10, 6981-7000) the present results constitute a complete assignment of all resonances of iminoprotons involved in the secondary structure of yeast tRNAPhe with a reliability and spectral resolution not reached heretofore. Separate identification of the methylprotons in m5C40 and m5C49 was also possible due to specific NOE patterns in the lowfield part of the spectrum. Our experiments indicate that in solution the psi 39 residue in the anticodon stem is orientated in a syn conformation in contrast to the normally observed anti orientation of the uracil base in AU basepairs. Evidence is presented that in solution the acceptor stem is stacked upon the T stem. Furthermore, it turns out that in a similar way the anticodon stem forms a continuous stack with the D stem, but here the m2(2)G26 residue is located between the latter two stems (as is found in the X-ray crystal structure). The stacking of these stems is not strictly dependent on the presence of magnesium ions. NOE experiments show that these structural features are preserved when proceeding from a buffer with magnesium ions to a buffer without magnesium ions although differences in chemical shifts and NOE intensities indicate changes in the conformation of the tRNA.     

Nuclear magnetic resonance studies on yeast tRNAPhe I. Assignment of the iminoproton resonances of the acceptor and D stem by means of Nuclear Overhauser Effect experiments at 500 MHz

Resonances of the water exchangeable iminoprotons of the acceptor and D stem of yeast tRNAPhe have been assigned by means of Nuclear Overhauser Effects (NOE's). Assignments were made for spectra recorded from tRNA dialysed against a buffer with 110 mM sodium and 5 mM magnesium ions and against a buffer with 430 mM sodium and no magnesium ions. Remarkable is the assignment of a resonance at 13.6 - 13.7 ppm to the iminoproton of C11G24. This assignment as well as those of G1C72, G3C70, U7A66, U12A23 and C13G22 are different from those made previously on the basis of less direct evidence. NOE experiments performed at 45 degrees C support the view that the D stem together with the tertiary interaction U8A14 is one of the most stable parts of the molecule in the presence of magnesium ions. A comparison of the spectra recorded under the two different buffer conditions shows that an excess of 320 mM sodium ions is not capable to force the tRNA in the same conformation as 5 mM magnesium ions can do.     

A nuclear magnetic resonance study of secondary and tertiary structure in yeast tRNAPhe.

We present experimental evidence which confirms recently proposed ring current prediction methods for assigning hydrogen-bond proton nuclear magnetic resonance (NMR) spectra from tRNA (Robillard, G. T., Tarr, C. E., Vosman, F., & Berendsen, H. J. C. (1976) Nature (London) 262, 363-369; Robillard, G. T., Tarr, C. E., Vosman, F., & Sussman, J. L. (1977) Biophys. Chem. 6, 291-298). The evidence is a series of temperature-dependent studies on yeast tRNAPhe monitoring both the high- and low-field NMR spectral regions, which are correlated with independent optical and temperature-jump (temp-jump) studies performed under identical ionic strength conditions. Using assignments derived from the new prediction methods, the melting patterns of the hydrogen-bonded resonances agree with those expected on the basis of optical, temp-jump, and NMR studies on the high-field spectral region. The implication of these results is that previous assignment procedures are at least partially incorrect and, therefore, studies based on those procedures must be reexamined.     

Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G1 X C72 and C2 X G71 should be reversed). The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.     

Epidermal growth factor from the mouse. Structural characterization by proton nuclear magnetic resonance and nuclear overhauser experiments at 500 MHz

Mouse epidermal growth factor (mEGF), a protein hormone effector molecule that regulates cellular development and division, has been investigated by using proton nuclear magnetic resonance techniques at 500 MHz. Well-resolved downfield aromatic and alpha-CH proton resonances (5.0-8.0 ppm) and an upfield ring current shifted isoleucine delta-methyl resonance (approximately 0.5 ppm) have been examined by using principally nuclear Overhauser methods. The data are analyzed in terms of model building based on the predictive Chou-Fasman secondary structure algorithm applied to mEGF [Holladay, L. A., Savage, C. R., Cohen, S., & Puett, D. (1976) Biochemistry 15, 2624-2633], which suggests the existence of some beta-structure and little or no alpha-helicity. Proximity relationships derived from nuclear Overhauser data among Tyr-3, -10, and -13, His-22, and Ile-23 allow refinement of some aspects of the predicted secondary structure and render additional information on how the protein backbone in mEGF is folded (i.e., tertiary structure). Nuclear Overhauser effects (NOEs) from irradiation of several alpha-CH proton resonances give evidence for tiered beta-sheet structure in mEGF. Such proximity relationships derived from NOE data place stringent limitations on possible models for the molecule. pH titration data demonstrate a His-22 pKa of 7.1, indicating either a salt bridge or hydrogen-bond formation between His-22 and another residue. The His-22 pKa is also reflected in the chemical shift changes of several other resonances as a function of pH. Nuclear Overhauser methods, used to differentiate direct (protonation) and indirect (conformation) effects on the chemical shift changes in the spectra of mEGF by varying the pH, yield evidence for a pH-induced conformational transition in the protein hormone associated with the breaking of the His-22 salt bridge or hydrogen bond.     

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.     

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.     

Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

The downfield aromatic (6-8 ppm) and upfield ring current shifted methyl regions (1-0 ppm) in the proton nuclear magnetic resonance spectrum of acyl carrier protein (ACP) from Escherichia coli have been examined at 500 MHz by using nuclear Overhauser methods. The data are analyzed in terms of the secondary structural model of Rock & Cronan (1979) [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785], which suggests the existence of four alpha-helical segments joined by three beta-turns, and a short coil at the C terminus of the protein. Nuclear Overhauser effects among Tyr-71, Ile-69, Ile-72, and His-75 allow refinement of the secondary structure of the C terminus. Nuclear Overhauser effects among Tyr-71, Phe-28, and three Ile's also place stringent limitations on the folding of the four alpha-helices. These data allow the proposal of a tertiary structural model for ACP.     

Nuclear magnetic resonance studies on transfer ribonucleic acid: assignment of AU tertiary resonances.

The hydrogen-bonded ring NH nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acid (RNA) species have been examined with particular emphasis on the extreme low-field portion. Betwen --13.8 and --15 ppm there are two extra resonances which are not derived from cloverleaf base pairs. A combined approach involving undermodified tRNAs, chemical modification, and hairpin fragment studies has assigned the T54--A58 resonance at --14.3 ppm in yeast tRNAPhe and Escherichia coli tRNA1 Val., the U8--A14 resonance has been assigned at --14.3 ppm, and the s4U8--A14 resonance in bacterial tRNAs has been assigned at --14.9 ppm. The T54--A58 resonance shifts between --14.3. and --13.8 ppm depending on the surrounding nucleotide sequence in the ribothymidine loop.     

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man₈GlcNAc₂Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.     

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectrum of a ribonuclease-resistant fragment of 5S ribonucleic acid

The downfield (9-15 ppm) proton NMR spectrum of a RNase A resistant fragment of E. coli 5S RNA has been studied by nuclear Overhauser methods. The fragment comprises bases 1-11 and 69-120 of the parent molecule [Douthwaite, S., Garrett, R.A., Wagner, R., & Feunteun, J. (1979) Nucleic Acids Res. 6, 2453-2470]. The nuclear Overhauser data identify two double helical structures in the fragment whose sequences are (GC)3(AU)(GC)3 and (GC)2(AU)(GU). These structures correspond exactly to the central portions of the terminal stem and procaryotic loop helices which should exist in the fragment sequences according to the Fox-Woese model [Fox, G.E., & Woese, C. R. (1975) Nature (London) 256, 505-506] of 5S RNA secondary structure. The significance of these and other nuclear Overhauser effects detected for the structure of 5S RNA and its fragment is discussed.     

Pulsed FT-NMR double resonance studies of yeast tRNAPhe: specific nuclear Overhauser effects and reinterpretation of low temperature relaxation data.

Cross-relaxation effects are demonstrated between the imino protons and other protons in yeast tRNAPhe and H2O. A detailed examination has been made of the observed relaxation rate of the proton resonance at 11.8 ppm from DSS as a function of the D2O content in the solvent. This result, as well as the size and number of observed nuclear Overhauser effects, suggests that dipolar magnetization transfer between solvent H2O, amino, imino, and other tRNA protons may dominate the relaxation processes of the imino protons at low temperature. At higher temperatures the observed relaxation rate is dominated by chemical exchange. The selective nuclear Overhauser effects are shown to be an important aid in resonance assignments. By these means we were able to identify tow protons from the wobble base pair GU4 at 11.8 ppm and 10.4 ppm.     

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.     

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25

The downfield (9-15 ppm) proton spectrum of Escherichia coli 5S RNA has been examined at 500 MHz by using nuclear Overhauser methods. The data confirm the existence of the terminal and procaryotic loop helices within the molecule [Fox, G. E., & Woese, C. R. (1975) Nature (London) 256, 505-506]. Very little stable, double-helical structure is detectable in the third loop of the molecule, the one comprising bases 12-68. The downfield spectrum of 5S RNA is perturbed in a highly specific manner upon addition of protein L25 to the system. The changes seen strongly suggest that the binding site for L25 on 5S RNA includes the procaryotic loop helix, but not the terminal stem helix. Similar complexes formed between L25 and the 5S RNA fragment consisting of bases 1-11, 69-87, and 89-120 show exactly the same spectral alterations. A number of downfield resonances appear in the spectra of these complexes which have no counterparts in the free RNA, suggesting the stabilization of new RNA structures by the protein. There are some indications of protein-nucleic acid nuclear Overhauser effects.     

Nuclear magnetic resonance observation of the triple interaction between A9 and AU12 in yeast tRNAPhe.

The nuclear Overhauser effect (NOE) was used to identify one of the amino proton resonances of base A23 in interaction with A9. These bases form a triple with U12 in the D stem of yeast tRNAPhe. The identification was verified by finding an NOE from this amino proton to the C8 proton of A9, as determined by comparisons of NOE's in a native and a C8-deuterated sample.     

Assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor homologue K from Dendroaspis polylepis polylepis. Two-dimensional nuclear magnetic resonance at 360 and 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the trypsin inhibitor homologue K from the venom of Dendroaspis polylepis polylepis is described and documented. The assignments are based entirely on the amino acid sequence and on 2-dimensional n.m.r. experiments at 360 and 500 M Hz. Individual assignments were obtained for the backbone and C beta protons of all 57 residues of the inhibitor homologue K, with the exceptions of the N-terminal amino group, the amide protons of Arg16, Gly37 and Gly40 and the C beta protons of Arg16 and Pro19. The assignments for the non-labile protons of the amino acid side-chains are complete, with the exception of Gln29, Glu49 and all the proline, lysine and arginine residues. For Asn and Trp the labile side-chain protons have also been assigned. The chemical shifts for the assigned resonances are listed for an aqueous solution at 50 degrees C and pH 3.4.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid.

The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.