Backbone Sites Research Articles

Reference energy extremal optimization: A stochastic search algorithm applied to computational protein design

We adapt a combinatorial optimization algorithm, extremal optimization (EO), for the search problem in computational protein design. This algorithm takes advantage of the knowledge of local energy information and systematically improves on the residues that have high local energies. Power-law probability distributions are used to select the backbone sites to be improved on and the rotamer choices to be changed to. We compare this method with simulated annealing (SA) and motivate and present an improved method, which we call reference energy extremal optimization (REEO). REEO uses reference energies to convert a problem with a structured local-energy profile to one with more random profile, and extremal optimization proves to be extremely efficient for the latter problem. We show in detail the large improvement we have achieved using REEO as compared to simulated annealing and discuss a number of other heuristics we have attempted to date.

Attosecond Electron Delocalization in the Conduction Band through the Phosphate Backbone of Genomic DNA

Partial density of states in the empty conduction band of the phosphate backbone sites in DNA was probed using energy-dependent resonant Auger spectroscopy. Results show that genomic DNA with periodic backbones exhibits an extended state despite separation of each phosphate group by an insulating sugar group. In antisense DNA with an aperiodic backbone, the equivalent state is localized. Remarkably rapid electron delocalization occurs at ca. 740 attoseconds for wet DNA, as estimated using the core-hole clock method. Such delocalization is comparable to the Fermi velocity of carbon nanotubes.

Triple resonance experiments for aligned sample solid-state NMR of 13C and 15N labeled proteins

Initial steps in the development of a suite of triple-resonance 1H/ 13C/ 15N solid-state NMR experiments applicable to aligned samples of 13C and 15N labeled proteins are described. The experiments take advantage of the opportunities for 13C detection without the need for homonuclear 13C/ 13C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ∼20% randomly labeled with 13C in all backbone and side chain carbon sites and ∼100% uniformly 15N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13C labeled at only the α-carbon and 15N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13C/ 13C decoupling while detecting 13C signals is avoided in the first case because of the low probability of any two 13C nuclei being bonded to each other; in the second case, the labeled 13C α sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13C chemical shift and 1H– 13C and 15N– 13C heteronuclear dipolar coupling frequencies associated with the 13C α and 13C′ backbone sites, which provide orientation constraints complementary to those derived from the 15N labeled amide backbone sites. 13C/ 13C spin-exchange experiments identify proximate carbon sites. The ability to measure 13C– 15N dipolar coupling frequencies and correlate 13C and 15N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13C chemical shift and 1H– 13C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.

Conformational Flexibility of a Microcrystalline Globular Protein: Order Parameters by Solid-State NMR Spectroscopy

The majority of protein structures are determined in the crystalline state, yet few methods exist for the characterization of dynamics for crystalline biomolecules. Solid-state NMR can be used to probe detailed dynamic information in crystalline biomolecules. Recent advances in high-resolution solid-state NMR have enabled the site-specific assignment of (13)C and (15)N nuclei in proteins. With the use of multidimensional separated-local-field experiments, we report the backbone and side chain conformational dynamics of ubiquitin, a globular microcrystalline protein. The measurements of molecular conformational order parameters are based on heteronuclear dipolar couplings, and they are correlated to assigned chemical shifts, to obtain a global perspective on the sub-microsecond dynamics in microcrystalline ubiquitin. A total of 38 Calpha, 35 Cbeta and multiple side chain unique order parameters are collected, and they reveal the high mobility of ubiquitin in the microcrystalline state. In general the side chains show elevated motion in comparison with the backbone sites. The data are compared to solution NMR order parameter measurements on ubiquitin. The SSNMR measurements are sensitive to motions on a broader time scale (low microsecond and faster) than solution NMR measurements (low nanosecond and faster), and the SSNMR order parameters are generally lower than the corresponding solution values. Unlike solution NMR relaxation-based order parameters, order parameters for (13)C(1)H(2) spin systems are readily measured from the powder line shape data. These results illustrate the potential for detailed, extensive, and site-specific dynamic studies of biopolymers by solid-state NMR.

Determinations of 15N Chemical Shift Anisotropy Magnitudes in a Uniformly 15N,13C-Labeled Microcrystalline Protein by Three-Dimensional Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C alpha, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C alpha and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between beta-sheet and alpha-helix residues are observed; the average value for the anisotropy parameter, delta (delta = delta(zz) - delta(iso)), for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues. The results show a variation in delta of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone phi torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.

The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation

Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4-dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, α-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed.

Site-Specific 13C Chemical Shift Anisotropy Measurements in a Uniformly 15N,13C-Labeled Microcrystalline Protein by 3D Magic-Angle Spinning NMR Spectroscopy

In this Communication, we introduce a 3D magic-angle spinning recoupling experiment that correlates chemical shift anisotropy (CSA) powder line shapes with two dimensions of site-resolved isotropic chemical shifts. The principal tensor elements from 127 ROCSA line shapes are reported, constraining 102 unique backbone and side-chain 13C sites in a microcrystalline protein (the 56 residue beta1 immunoglobulin binding domain of protein G). The tensor elements, determined by fitting to numerical simulations, agree well with quantum chemical predictions. The experiments, therefore, validate calculations of CSAs in a protein of known structure. The data will be useful for the development of side-chain CSA quantum calculations and will aid in the design and interpretation of solution NMR experiments that utilize CSA-dipole cross-correlation to constrain torsion angles or to enhance resolution and sensitivity (such as in TROSY). Furthermore, the methodology described here will enable databases of CSA data to be generated with higher efficiency, for purposes of direct protein structure refinement.

Detection of saponins in extract of Panax notoginseng by liquid chromatography–electrospray ionisation-mass spectrometry

The liquid chromatography–electrospray ionisation-mass spectrometry (LC–ESI-MS) method was developed for the analyses and identification of saponins in plant extract from the root of Panax notoginseng (Burk.) F.H. Chen. The HPLC experiments were proceeded by means of a reversed-phase C18 column and a binary mobile phase system consisting of 0.2% acetic acid and acetonitrile under gradient elution conditions. Eight major peaks were separated and detected using both evaporative light scattering and MS detectors. The mass spectrometer was operated in the negative ion mode using electrospray ionization. The molecular ions, [ M − H] − and the adduct ions [ M + AcO] − of saponins were observed, and from which the molecular weights were obtained. A collision-induced dissociation (CID) experiment was carried out to aid the identification of the backbone and glycosidic linkage sites of the saponins. The identification of the saponins (peaks 1–7) in the extract of P. notoginseng was based on matching their retention times, the detection of the saponin molecular ions, and the fragment ions of the molecular ion obtained in the CID experiments with those of the authentic standards and data reported in the literature. The molecular structure of peak 8 was elucidated according to the fragmentation patterns and the literature reports.

19F and 13C NMR Signal Assignment and Analysis in a Perfluorinated Ionomer (Nafion) by Two-Dimensional Solid-State NMR

The 19F and 13C NMR resonances of the perfluorinated ionomer, Nafion, are assigned to their corresponding chemical groups using two-dimensional (2D) 13C−19F heteronuclear correlation and 19F-exchange NMR experiments under 28 or 30 kHz magic-angle spinning, combined with peak area and relaxation time information. On the basis of these new experimental data, we revise the assignment of more than half of the resolved 19F NMR peaks. In particular, the backbone CF group is shown to resonate at −138 ppm, the side-group CF at −144 ppm, and the SCF2 group, which can be selected by a T2 filter, at −117 ppm. The OCF2 groups resonate slightly downfield from the CF3 fluorines. Deconvolution of the 19F and 13C spectra based on cross sections from the 2D spectra provides the peak widths and positions of all side group and several backbone sites. The inhomogeneous broadening observed in both 13C and 19F NMR spectra for the sites near the backbone CF group reveals static disorder near the branch point, which contrasts with the high conformational order of the rest of the backbone and the mobility of the ends of the side group.

Solid-state NMR spin diffusion for measurement of membrane-bound peptide structure: gramicidin A.

A recently developed solid-state NMR method for measurement of depths in membrane systems is applied to gramicidin A, a membrane-bound peptide of known structure, to investigate the potential of this method. (15)N-detected, (1)H spin diffusion experiments demonstrate the resolution of the technique by measuring the 4-5 A depth differences between three (15)N-labeled backbone sites (Trp13, Val7, Gly2) in gramicidin A. We also show that (13)C-detected, (1)H spin diffusion experiments on unlabeled gramicidin A are sufficient to discriminate between the end-to-end dimer and double-helix structures of gramicidin A. Thus, spin diffusion solid-state NMR experiments can provide a simple approach, which does not require labeled samples, for testing structural models of membrane-bound peptides.

Supramolecular Metal—Organometallic Coordination Networks Based on Quinonoid π‐Complexes

AbstractFor Abstract see ChemInform Abstract in Full Text.

Self‐Assembly of Metal‐Organometallic Coordination Networks

AbstractFor Abstract see ChemInform Abstract in Full Text.

Supramolecular metal-organometallic coordination networks based on quinonoid pi-complexes.

The use of organometallic pi-complexes in the coordination-directed self-assembly of polymeric structures is a new area with many potential applications. Supramolecular metal-organometallic coordination networks (MOMNs), which are described herein, consist of metal ion or metal cluster nodes connected by bifunctional "organometalloligands" that serve as spacers. The organometalloligand utilized in this work is the stable anionic complex (eta(4)-benzoquinone)Mn(CO)(3)(-) (p-QMTC), which binds through both quinone oxygen atoms to generate MOMNs having both backbone and pendant metal sites. In many cases the MOMNs are obtained as neutral and thermally stable solids, with molecular structures that depend on the geometrical and electronic requirements of the metal nodes, the solvent, and the presence of organic spacers. Redox-active quinone-based organometallic pi-complexes permit the construction of an impressive range of coordination network architectures and hold much promise for the development of functional materials.

Self‐assembly of metal‐organometallic coordination networks

AbstractA range of neutral metal‐organometallic coordination networks (MOMNs) containing both backbone and pendant metal sites have been synthesized and characterized. These materials consist of metal ions or metal ion clusters as nodes that are linked by the bifunctional “organometalloligand” (η4‐benzoquinone)Mn(CO)3−, which binds through the quinone oxygen atoms. The resulting MOMNs can be rationally designed to a significant extent based upon a knowledge of the electronic and geometrical requirements of the metal ion nodes, the solvent, organometalloligand substituents, and the presence or absence of organic spacers. An impressive range of architectures have been accessed in this manner, suggesting that the use of π‐organometallics in coordination directed self‐assembly holds much promise.

Poly[p-(phenyleneethynylene)-alt-(thienyleneethynylene)] Polymers with Oligopyridine Pendant Groups: Highly Sensitive Chemosensors for Transition Metal Ions

We report the synthesis and characterization of a series of luminescent chemosensory materials that have the conjugated polymer poly[p-(phenyleneethynylene)-alt-(thienyleneethynylene)] (PPETE) as a backbone and oligopyridine pendant sites as receptors. These polymers are soluble in common organic solvents and highly emissive. Investigations reveal that these polymers are highly sensitive to transition metal ions such as Ni2+. For the terpyridine−receptor polymer ttp−PPETE, we observed ∼5% initial emission quenching by Ni2+ concentration as low as 4 × 10-9 M. The polymers also show selectivity to different transition metal ions. The ether-linked polymer ttp−O−PPETE is also prepared which successfully demonstrates that complete conjugation is not required for emission quenching. However, the vinylene-linked ttp−PPETE was found to have a higher quenching efficiency.

Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptic to yield complementary sequence information.

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.

Two- and Three-Dimensional 1H/13C PISEMA Experiments and Their Application to Backbone and Side Chain Sites of Amino Acids and Peptides

Two-dimensional 1H/13C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the 1H–13Cα dipolar coupling frequency with the chemical shift frequencies of the α-carbon, as well as the directly bonded amide 15N site, is also demonstrated. In this experiment the large 1H–13Cα heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping 13C resonances of oriented polypeptides, while simultaneously providing measurements of the 13Cα chemical shift, 1H–13C dipolar coupling, and 15N chemical shift frequencies and angular restraints for backbone structure determination.

Molecular Dynamics Simulation in Solvent of the Bacteriophage 434 cI Repressor Protein DNA Binding Domain Amino Acids (R1–69) in Complex with its Cognate Operator (OR1) DNA Sequence

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the 434 cl Repressor protein DNA binding domain (DBD) amino acids (R1–69) and DNA of operator (OR1) and its flanks consisting of 28 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the 434 cl repressor DNA recognition helix 3 formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ORl DNA major groove halfsites and flanking regions. In addition, hydrophilic amino acids within the loop between helix 3 and helix 4 have strong electrostatic attraction to codon-anticodon nucleotides located within the central nucleotides of the minor groove between the ORI major groove halfsites These interactions together induced significant structural changes in the operator DNA manifested by overtwisting of the central nucleotide base pairs and narrowing of the minor groove between the DNA major groove halfsites. Finally, these findings offer a code for site specific DNA recognition by the 434 cl repressor protein.

Three-Dimensional 13C Shift/1H–15N Coupling/15N Shift Solid-State NMR Correlation Spectroscopy

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee–Goldburg irradiation for both 13C homonuclear decoupling and 1H–15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H–15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.

Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process

Neutralization-reionization mass spectrometry (MS)1 is of unique value for preparing and characterizing highly reactive and unstable neutral species, such as the intermediate in the dissociative-recombination reaction H3O + ef H2O + H + 6.4 eV.2 Following an earlier suggestion,3 using neutralization accompanying surface-induced dissociation (SID)4 to form an unstable site did not yield new cleavage reactions5 in multiply charged protein cations from electrospray ionization (ESI) with Fourier transform (FT) MS.6 Serendipitously, we now find that one or more charges on such protein cations can be neutralized with low-energy electrons to cause specific cleavage of the amine bond to form c, z products,7 in contrast to the amide cleavage b, y products formed by collisionally activated dissociation (CAD),8 infrared multiphoton (IRMPD)9 and UV10 photodissociation, 70 eV electron impact excitation,11 and SID.5 The b, y products are formed by the lowest energy backbone cleavage of ESI protein ions.6-9 An attempt to cleave stronger bonds using high-energy (6.4 eV) 193 nm photons gave mainly b, y products for 2 kDa protein ions,10 but for 2.8 and 8.6 kDa protein ions12 gave small yields of c, z amine bond cleavage products not previously observed. In this further investigation, extra electrodes were placed outside the ion cell electrodes that trap the positively charged ions. With the outside electrodes at +9 V,13 extensive 193 nm laser irradiation of SWIFT-selected14 (M + 11H)11+ ubiquitin ions (8.6 kDa) only produces b, y, not c, z, ions. However, with the outside electrodes at -1 V, the c, z products are formed along with 10+ molecular ions; unexpectedly, these are mainly (M + 11H)10+• ions (Figure 1d), 1 Da heavier than the (M + 10H)10+ ions formed by ESI (Figure 1c). The 4+ mellitin ion spectrum measured under the same conditions (Figure 1b) similarly contains (M + 4H)3+•, consistent with capture of secondary electrons formed by the 193 nm photons impinging on metal surfaces and trapped by the -1 V electrodes: (M + 4H)4+ + ef (M + 4H)3+•.15 Electrons were produced instead (no laser) by a conventional heated filament source outside the FTMS magnet opposite the ESI source.11 With a 10-5 Torr Ar pulse for ecooling (energy < 0.2 eV; an SF6 pulse lowered the efficiency), the 11+ ions of ubiquitin gave a spectrum that showed c, z cleavage of 50 out of 75 backbone positions; CAD8/IRMPD9 gave b, y cleavage of eight of these positions plus seven others. Cooled electrons plus the 15+ ions of FeIII equine cytochrome c16 produced (Figure 2) c, z fragment ions from cleavages at all but 40 of the 103 possible backbone sites (e.g., N-terminal side of Pro, none; of Ile, Leu, Val, few); CAD produces b, y cleavages (total 19) at eight additional sites. The 21+ apomyoglobin ions (17 kDa) yielded 33 c, z cleavages, but the 34+ ions of bovine carbonic anhydrase (29 kDa) as yet has given only 33+, 32+, and 31+ molecular ions. Electron capture dissociation (ECD)1-3 rationalizes these results. The capture cross section should be proportional to the ionic charge squared, consistent with the minimal secondary fragmentations to produce internal ions and the predominance of cleavages in the central∼70% of the protein chain. Charge values and masses17 of the complementary product ions are consistent with dissociation after ecapture, such as c39/z37 from the 76-residue ubiquitin 11+ ions and c69/z35 from the 104residue cytochrome c 15+ ions. The most favored protonation sites are the side chains of Lys, Arg, and His;18 neutralization to form hypervalent species1-3 at Lys and Arg would account for ions (Figure 2) representing losses of 17, 44, and 59 Da from (M + nH)(n-x)+ (eq 1; neutralization of protonated His gives a more stable radical site).