ALBERT

Notes: Individual hydration water molecules in aqueous protein solutions have been observed using experimental schemes for homonuclear two-dimensional and heteronuclear three-dimensional NMR experiments in H2O solution, which do not require suppression of the solvent line by presaturation. In these experiments, the location of the hydration waters is determined from their nuclear Overhauser effects (NOEs) with individual hydrogen atoms of distinct amino acid residues. In the basic pancreatic trypsin inhibitor (BPTI), four internal water molecules that had been reported in three different crystal forms were also found to be in the same locations in the solution structure, with lifetimes with respect to exchange of the water protons in excess of 0.3 ns. Additional NOEs with polypeptide protons located on the protein surface may involve either hydration water molecules or hydroxyl protons of amino acid side chains. Their total number is small compared to the number of NOEs expected from the hydration water molecules identified in the crystal structures of BPTI.

Notes: Why Pentose- and Not Hexose-Nucleid Acids? Part IV . ‘Homo-DNA’: 1H-, 13C-, 31P-, and 15N-NMR-Spectroscopic Investigation of ddGlc(A-A-A-A-A-T-T-T-T-T) in Aqueous SolutionFrom a comprehensive NMR structure analysis, it is concluded that the ‘homo-DNA’ oligonucleotide ddGlc(A-A-A-A-A-T-T-T-T-T) in 3 mM D2O solution (100 mM NaCl, 50 mM phosphate buffer, pH 7.0, T = 50°) forms a duplex of C2-symmetry, with its self-complementary oligonucleotide strands in antiparallel orientation. The 2′,3′-dideoxy-β-D-glucopyranosyl rings are in their most stable chair conformation, with all three substituents equatorial and with the adenine as well as the thymine bases in the anti-conformation. The base pairing is of the Watson-Crick type; this pairing mode (as opposed to the reverse-Hoogsteen mode) was deduced from the observation of inter strand NOEs between the adenine protons H—C(2) and the pyranose protons Hα-C(2′) of the sequentially succeeding thymidine nucleotides of the opposite strand, a correlation which discriminates between the Watson-Crick and the reverse-Hoogsteen pairing mode. The NOEs of the NH protons with either the adenine protons H—C(2) or H—C(8), that are normally used to identify the pairing mode in DNA duplexes, cannot be observed here, because the NH signals are very broad. This line broadening is primarily due to the fact that the exchange of the imino protons with the solvent is faster than for corresponding DNA duplexes.Computer-assisted modeling of the [ddGlc(A5-T5)]2 duplex with the program CONFOR [23], using the linear (idealized) homo-DNA single-strand conformation (α = -60°, β = 180°, γ = 60°, δ = 60°, ∊ = 180°, ζ = -60°, see [1] [3]) as the starting structure, resulted in two duplex models A and B (see Figs. 27-32, Scheme 9, and Table 4) which both contain quasi-linear double strands with the base-pairing axis inclined relative to the strand axes by ca. 60° and 45°, respectively, and with base-pair stacking distances of ca. 4.5 Å. While neither of the two models, taken separately, can satisfy all of the NMR constraints, the NMR data can be rationalized by the assumption that the observed duplex structure represents a dynamic equilibrium among conformers which relate to models A and B as their limiting structure. The required rapid equilibrium appears feasible, since the models A and B are interconvertible by two complementary 120° counter rotations around the α-axis and the γ-axis, respectively, of the phosphodiester backbone. The models A and B correspond to the two types of linear (idealized) single-strand backbone conformation derived previously by qualitative conformational analysis without and with allowance for gauche-trans-phosphodiester conformations, respectively [1] [3]. Refinement of the models A and B with the use of the program AMBER [27] by energy minimization in a water bath and molecular-dynamics simulations (2 ps, 300° K) resulted in two dynamic structures (Figs. 33 and 34, Table 4). These have roughly the same energy, closely resemble the starting structures A and B, and satisfy - as an ensemble - all of the NMR constraints without violating any van der Waals distances by more than 0.2 Å. Extensive fluctuations in base-pair distance and deviations from base-pair coplanarity, as well as the presence of water molecules in the cavities between some of the base pairs, were observed in both dynamic structures A and B, which, on the other hand, did not mutually interconvert within the short simulation time period used. These model properties, together with the conjectured equilibrium between the two structure types A and B, lead to the hypothesis of a homo-DNA duplex containing a ‘partially molten’ pairing core. This proposal could qualitatively account for a high rate of the NH exchange, as well as for part of the previously established [3] deficits in both enthalpic stabilization and entropic destabilization of homo-DNA duplexes relative to corresponding DNA duplexes. The phenomenon of the higher overall stability of homo-DNA duplexes vs. DNA duplexes (e.g, [ddGlc(A5-T5)]2, Tm = 59° vs. [d(A5-T5)]2, Tm = 33°, both at c ≈ 50 μM [3]) can then be seen as the result not only of a higher degree of conformational preorganization of the homo-DNA single strand toward the conformation of the duplex backbone [1] [3], but also of the entropic benefit of greater disorder in the central pairing zone of the homo-DNA duplex. This view of the structure of a homo-DNA duplex relates its characteristic properties to a central structural feature: the average base-pair distance in the models of homo-DNA is too large for regular base stacking (ca. 4.5 Å vs. ca. 3.5 Å in DNA). This difference in the distances between adjacent base pairs is a direct consequence of the quasi-linearity of the homo-DNA double strand as opposed to the right-handed twist of the helical DNA duplexes [1] [3], which is directly related to the specific conformational properties of pyranose rings as opposed to furanose rings [1]. Thus, the structural hypothesis derived from the NMR analysis of [ddGlc(A5-T5)]2 relates the conformational differences between homo-DNA and DNA directly to the sugar ring size, which is the essential constitutional difference between the two types of structure.The English footnotes to Figs. 1-34, Schemes 1-9, and Tables 1-4 provide an extension of this summary.

Notes: A new program for automatic resonance assignment of nuclear magnetic resonance (NMR) spectra of proteins, GARANT (General Algorithm for Resonance Assignment), is introduced. Three principal elements used in this approach are: (a) representation of resonance assignments as an optimal match of two graphs describing, respectively, peaks expected from combined knowledge of the primary structure and the magnetization transfer pathways in the spectra used, and experimentally observed peaks; (b) a scoring scheme able to distinguish between correct and incorrect resonance assignments; and (c) combination of an evolutionary algorithm with a local optimization routine. The score that evaluates the match of expected peaks to observed peaks relies on the agreement of the information available about these peaks, most prominently, but not exclusively, the chemical shifts. Tests show that the combination of an evolutionary algorithm and a local optimization routine yields results that are clearly superior to those obtained when using either of the two techniques separately in the search for the correct assignments. GARANT is laid out for assignment problems involving peaks observed in two- and three-dimensional homonuclear and heteronuclear NMR spectra of proteins. © 1997 by John Wiley & Sons, Inc.

Notes: The longitudinal 13C spin relaxation times T1 and the 13C{1H} nuclear Overhauser enhancement were measured in a concentrated aqueous solution of the basic pancreatic trypsin inhibitor. The correlation time for overall rotational motions of the basic pancreatic trypsin inhibitor molecules was found to be τR ≈ 2 × 10-8 s. In connection with previous 1H n.m.r. studies of intramolecular motions of the aromatics, we were particularly interested in the correlation times τG for intramolecular segmental motions of the aromatic rings. The present experiments revealed no manifestation of intramolecular motions of the aromatics, indicating that τG ≫ 2 × 10-8 s for the aromatic ring carbon atoms. On the other hand, rapid segmental motions were evidenced for the peripheral carbon atoms of aliphatic amino acid sidechains. Comparison of the 1H and 13C n.m.r. data on the basic pancreatic trypsin inhibitor indicates that the time scale of high resolution 1H n.m.r. at high fields may in many instances be more appropriate for studies of the molecular dynamics in globular proteins than the time scale of spin relaxation measurements.

Notes: The potential of high resolution nuclear magnetic resonance (NMR) for many-parameter characterization of biopolymer conformation has long been recognized. Currently new NMR techniques for use in this field are in full development. In the present paper a survey of fundamental aspects of NMR studies of protein conformation is used as a basis for discussion of recently developed one-dimensional and two-dimensional NMR experiments for studies of proteins.

Notes: 1H-NMR. studies of the protected linear tetrapeptides CF3CO-Gly-Gly-L-Tyr-L-Ala-OCH3, CF3CO-Gly-L-Ala-L-Tyr-L-Ala-OCH3 and CF3CO-Gly-L-Ala-L-Tyr-Gly-OCH3 showed that the side chain of the tyrosyl residue was in all three peptides preferentially oriented towards the amino terminus of the peptide chain. This preferred spatial arrangement of the aromatic side chain was manifested in the chemical shifts of the amino acid residue preceding tyrosine and in the vicinal spin-spin coupling constants 3JHCαCβH of tyrosine.

Notes: Synthesis of Deuteriated Derivatives of Diastereomeric Cyclopentapeptides for Use in Conformational Analysis.The syntheses and properties of eight cyclic pentapeptides containing glycine with two deuterium atoms at C(α) (Gly*) are described: cyclo-[-Gly-L-Cys(DPM)-Gly-Gly*-L-Pro-], cyclo-[-Gly*-L-Cys(DPM)-Gly-Gly*-L-Pro-], cyclo-[-Gly-L-Cys-(DPM)-Gly-Gly*-D-Pro-], cyclo-[-Gly*-L-Cys(DPM)-Gly-Gly*-D-Pro-], cyclo-[-Gly-L-Ala-Gly-Gly*-L-Pro-], cyclo-[-Gly*-L-Ala-Gly-Gly*-L-Pro-], cyclo-[-Gly-L-Ala-Gly-Gly*-D-Pro-], and cyclo-[-Gly*-L-Ala-Gly-Gly*-D-Pro-] (DPM = diphenylmethyl). These particular compounds were chosen in order to assign by NMR. unambiguously certain conformational features, like hydrogen-bonding and shielding, to individual peptide bonds.

Notes: The 13C-NMR. in Zn(II)(Porphin), Fe(III)(Porphin)(CN2), Zn(II)(Tetraphenyl-porphin), and Fe(III)(Tetraphenylporphin)(CN2) have been identified, and the 13C hyperfine shifts in the iron complexes evaluated. It was found that dipole-dipole coupling with the electron spin localized in the π-orbitals of the aromatic carbon atoms makes an important contribution, to the 13C hyperfine shifts. In a preliminary analysis the experimental spin density distribution obtained from the combined 1H- and 13C-NMR.-data is compared with theoretical models of the iron porphyrin complexes.

Notes: The 13C-NMR. in Zn(II) (Protoporphyrin IX), Fe(III) (Protoporphrin IX) (CN)2, Zn(II) (Deuteroporphyrin IX dimethylester), and Fe(III) (Deuteroporphyrin IX) (CN)2 have been identified, and the 13C hyperfine shifts in the iron complexes evaluated. In a partial analysis of these data the parameters QCCH3H, QCCHH, and QC′CHC, which characterize the isotropic coupling through hyperconjugation between the 1H and 13C nuclei of the porphyrin side chains and the unpaired electron spin density on the aromatic ring carbon atoms, have been re-examined. This paper is part of an investigation of the electronic states in low spin ferric hemes and hemoproteins, and the relations between the electronic structures and the biological roles of these molecules.

Notes: The 1H- and 13C-NMR. spectra of cyclo-tetraglycyl show that the four peptide groups are magnetically equivalent, and different from either a standard trans or a standard cis peptide group. It is suggested that the observed NMR. features correspond to a non-planar form of the peptide groups. On the one hand these data confirm the earlier conclusions from theoretical investigations of the molecular geometry, that cyclic tetrapeptides could not contain four standard trans peptide groups. On the other hand they are not consistent with a previously suggested alternative molecular conformation according to which cyclo-tetraglycyl would adopt a conformation similar to cyclo-tetrasarcosyl, with two cis and two trans peptide bonds. The different behaviour of glycine and sarcosine under the steric strains of tetrapeptide ring closure would appear to suggest that with the exception of the X-Pro bonds, transoid peptide groups in polypeptide chains of the common amino acids should be more likely to occur than the cis form, which has as yet apparently not been observed for N-unsubstituted peptide groups in natural peptides or proteins.