Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination

X-ray crystallography and NMR spectroscopy provide the only sources of experimental data from which protein structures can be analyzed at high or even atomic resolution. The degree to which these methods complement each other as sources of structural knowledge is a matter of debate; it is often proposed that small proteins yielding high quality, readily analyzed NMR spectra are a subset of those that readily yield strongly diffracting crystals. We have examined the correlation between NMR spectral quality and success in structure determination by X-ray crystallography for 159 prokaryotic and eukaryotic proteins, prescreened to avoid proteins providing polydisperse and/or aggregated samples. This study demonstrates that, across this protein sample set, the quality of a protein's [15N-1H]-heteronuclear correlation (HSQC) spectrum recorded under conditions generally suitable for 3D structure determination by NMR, a key predictor of the ability to determine a structure by NMR, is not correlated with successful crystallization and structure determination by X-ray crystallography. These results, together with similar results of an independent study presented in the accompanying paper (Yee, et al., J. Am. Chem. Soc., accompanying paper), demonstrate that X-ray crystallography and NMR often provide complementary sources of structural data and that both methods are required in order to optimize success for as many targets as possible in large-scale structural proteomics efforts.     

Synthesis of an elusive,stable 2-azaallyl radical guided by electrochemical and reactivity studies of 2-azaallyl anions

The super electron donor (SED) ability of 2-azaallyl anions has recently been discovered and applied to diverse reactivity, including transition metal-free cross-coupling and dehydrogenative cross-coupling processes. Surprisingly, the redox properties of 2-azaallyl anions and radicals have been rarely studied. Understanding the chemistry of elusive species is the key to further development. Electrochemical analysis of phenyl substituted 2-azaallyl anions revealed an oxidation wave at E_1/2 or E_pa = −1.6 V versus Fc/Fc⁺, which is ∼800 mV less than the reduction potential predicted (E_pa = −2.4 V vs. Fc/Fc⁺) based on reactivity studies. Investigation of the kinetics of electron transfer revealed reorganization energies an order of magnitude lower than commonly employed SEDs. The electrochemical study enabled the synthetic design of the first stable, acyclic 2-azaallyl radical. These results indicate that the reorganization energy should be an important design consideration for the development of more potent organic reductants.

The super electron donor (SED) capabilities of 2-azaallyl anions has recently been discovered and applied to diverse reactivity; their structures and electron transfer characteristics are reported herein.     

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.     

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

N−H Bond Formation at a Diiron Bridging Nitride

Despite their connection to ammonia synthesis, little is known about the ability of iron-bound, bridging nitrides to form N−H bonds. Herein we report a linear diiron bridging nitride complex supported by a redox-active macrocycle. The unique ability of the ligand scaffold to adapt to the geometric preference of the bridging species was found to facilitate the formation of N−H bonds via proton-coupled electron transfer to generate a μ-amide product. The structurally analogous μ-silyl- and μ-borylamide complexes were shown to form from the net insertion of the nitride into the E−H bonds (E=B, Si). Protonation of the parent bridging amide produced ammonia in high yield, and treatment of the nitride with PhSH was found to liberate NH₃ in high yield through a reaction that engages the redox-activity of the ligand during PCET.     

Randomized k-server algorithms for growth-rate bounded graphs

The k-server problem is a fundamental online problem where k mobile servers should be scheduled to answer a sequence of requests for points in a metric space as to minimize the total movement cost. While the deterministic competitive ratio is at least k, randomized k-server algorithms have the potential of reaching o(k)-competitive ratios. Prior to this work only few specific cases of this problem were solved. For arbitrary metric spaces, this goal may be approached by using probabilistic metric approximation techniques. This paper gives the first results in this direction, obtaining o(k)-competitive ratio for a natural class of metric spaces, including d-dimensional grids, and wide range of k.