One- and two-dimensional NMR spectral analysis of the consequences of single amino acid replacements in proteins

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple-quantum and two-dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence-structure and structure-function relationships in proteins. Our NMR results provide examples of sequence effects on pKa' values, average conformation, and internal motion of amino acid side chains.     

Physiological studies onMarphysa gravelyi VII. Tissue respiration

Summary 1. The respiratory rate of body tissues of the polychaeteM. gravelyi ranged from 3.13 µl/mg/hr to 6.34 µl/mg/hr in the four experimental salinities 10 , 14, 17 and 24 Since similar estimations in other polychaetes are not available in literature a comparison is difficult. However, the trends, i. e., increase of respiratory rates with increasing salinity, are similar to those reported for whole individuals ofN. diversicolor.2. InM. gravelyi, both the organic and inorganic ions are regulated. It is argued that the trend reported above may be traced to the operation of an effective ion transport system.3. Two adaptations that may help to understand the trend of evolution inM. gravelyi are the reduction in permeability and the lowering of the body fluid concentration.4. These two adaptations may indicate a tendency towards life in freshwater. A few other features, such as corresponding changes in the reproductive patterns ofM. gravelyi and the geographical distribution of the genusMarphysa support this assumption.

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Physiologische Untersuchungen anMarphysa gravelyi. VII. Gewebsatmung

Kurzfassung Die respiratorische Aktivität des Körpergewebes vonMarphysa gravelyi Southern, einem brackwasserlebenden Polychaeten, wurde in verschiedenen Salinitätsstufen (10, 14, 17 und 24 S) gemessen. Es wurde festgestellt, daß mit zunehmendem Salzgehalt des Mediums der Sauerstoffverbrauch steigt. Diese Zunahme der Atmungsgröße wird in Beziehung zu einer verstärkten Ionenregulation gesetzt. Auf Grund osmoregulatorischer Befunde wird angenommen, daß bei dieser Art Tendenzen zur Anpassung an eine Existenz im Süßwasser bestehen.

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Dedicated to Professor Dr.Friedrich Krüger on his 65^th birthday, August 18, 1967.     

Distinct Ca2+ binding properties of novel C2 domains of plant phospholipase dalpha and beta

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDalpha requires millimolar levels of Ca(2+) for optimal activity, whereas PLDbeta is most active at micromolar concentrations of Ca(2+). Multiple amino acid sequence alignments suggest that PLDalpha and PLDbeta both contain a Ca(2+)-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDalpha and PLDbeta, designated PLDalpha C2 and PLDbeta C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major beta-sheet structures but underwent spectral changes upon binding Ca(2+); the magnitude was larger for PLDbeta C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca(2+) concentrations for PLDbeta C2 but at millimolar levels of the cation for PLDalpha C2. PLDbeta C2 exhibited three Ca(2+)-binding sites: one with a dissociation constant (K(d)) of 0.8 microm and the other two with a K(d) of 24 micrometer. In contrast, isothermal titration calorimetry data of PLDalpha C2 were consistent with 1-3 low affinity Ca(2+)-binding sites with K(d) in the range of 590-470 micrometer. The thermodynamics of Ca(2+) binding markedly differed for the two C2 domains. Likewise, PLDbeta C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca(2+) concentrations, whereas PLDalpha C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca(2+) displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca(2+) and PC binding properties of PLDalpha C2 and PLDbeta C2 follow a trend similar to the Ca(2+) requirements of the whole enzymes, PLDalpha and PLDbeta, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDalpha and PLDbeta have novel structural features and serve as handles by which Ca(2+) differentially regulates the activities of the isoforms.     

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Hydrogen-1 nuclear magnetic resonance investigation of Clostridium pasteurianum rubredoxin: previously unobserved signals

Previously unobserved signals were located in the 470-MHz 1H NMR spectra of oxidized and reduced rubredoxin (Rd) from Clostridium pasteurianum. When the protein was oxidized, some of the resonances broadened beyond detection. Longitudinal relaxation (T1) measurements identified a number of these peaks as arising from residues close to the paramagnetic iron; these resonances exhibited short T1 values attributable to the dominant electron-nuclear dipolar relaxation mechanism. The chemical shifts of these peaks were not strongly dependent on the oxidation state of the protein, although relative ratios of line widths of several peaks in the spectra of oxidized and reduced Rd suggested localized conformational changes of the protein as a result of oxidation. Furthermore, spectra of the oxidized protein collected in the range 8-60 degrees C revealed no appreciable changes in the chemical shifts of these peaks with temperature. These results seem to point out a negligible dipolar contribution, due to either magnetic anisotropy or zero field splitting, to the observed shifts in the spectrum of oxidized Rd. Resonances were assigned to tyrosine-11 or phenylalanine-49 (but not to either specifically) on the basis of their T1 values and the X-ray diffraction data of the protein molecule [Watenpaugh, K. D., Sieker, L. C., Herriott, J. R., & Jensen, L. H. (1973) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B29, 943-956; and a further refinement deposited with the Protein Data Bank]. An upfield-shifted peak at about -1.1 ppm in the spectra of both oxidized and reduced Rd was assigned to a methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)