Nuclear magnetic resonance captures the elusive

Innovative nuclear magnetic resonance approaches have emerged as outstanding means of tracking down functionally important properties of biomolecular assemblies that evade detection. These enigmatic attributes include dynamic equilibria with very lightly populated excited states and structures of complexes obscured by slow tumbling in solution

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Molecular genetic manipulation and characterization of the high-potential chain of cyclic electron transport in Rhodobacter sphaeroides

The Rieske iron-sulfur subunit of the cytochrome bc\sb{1} complex of Rhodobacter sphaeroides assembles in the E. coli and R. sphaerodies membranes with its iron-sulfur cluster intact, in the absence of the cytochrome subunits. This assembly, in light of predicted structural features of the hydrophobic amino-terminus and proteolytic release of a soluble domain, suggests the subunit may be anchored to the membrane by a single amino-terminal, transmembrane helix. Sequence homology with dioxygenase subunits binding a spectrally similar 2FE-2S cluster, suggests the fully conserved residues of the cytochrome bc\sb{1} and b\sb{6}f sequences ligating the iron-sulfur cluster may be the first and third cysteines and both histidines. When Gly133, neighboring the ligands of the cluster, is replaced by aspartate, quinol oxidation is specifically impaired at the Q\sb{\rm o} site.The heme-binding domain of cytochrome c\sb{1} released from the membrane to the periplasmic space by genetic removal of the carboxyl-terminal anchor is capable of reducing cytochrome c\sb{2} at a near normal rate. Truncation of cytochrome c\sb{1} causes a drop in its midpoint potential of 40 mV. The crippled complex remaining in the membrane lacking both the low potential b heme and cytochrome c\sb{1} can still bind stigmatellin and antimycin.Electron transport assay of a R. sphaeroides strain with cytochrome c\sb{2} genetically deleted shows that cytochrome c\sb{2} is essential to connect the cytochrome bc\sb{1} complex with the reaction center. A soluble c cytochrome, induced by second site mutations in the mutant, restores electron transfer between the cytochrome bc\sb1 complex and the reaction center.The usefulness of the alkaline phosphatase gene fusion assay of membrane protein topology is confirmed using the Rhodobacter sphaeroides reaction center subunit L. The fusions with junctions expected to lie near the periplasmic surface, based on the crystallographic structure of the reaction center, have high activity. These results suggest the assay can be applied to some membrane proteins foreign to E. coli.U of I OnlyETDs are only available to UIUC Users without author permissio

Binding Isotherms and Time Courses Readily from Magnetic Resonance

Evidence is presented that binding isotherms, simple or biphasic, can be extracted directly from noninterpreted, complex 2D NMR spectra using principal component analysis (PCA) to reveal the largest trend(s) across the series. This approach renders peak picking unnecessary for tracking population changes. In 1:1 binding, the first principal component captures the binding isotherm from NMR-detected titrations in fast, slow, and even intermediate and mixed exchange regimes, as illustrated for phospholigand associations with proteins. Although the sigmoidal shifts and line broadening of intermediate exchange distorts binding isotherms constructed conventionally, applying PCA directly to these spectra along with Pareto scaling overcomes the distortion. Applying PCA to time-domain NMR data also yields binding isotherms from titrations in fast or slow exchange. The algorithm readily extracts from magnetic resonance imaging movie time courses such as breathing and heart rate in chest imaging. Similarly, two-step binding processes detected by NMR are easily captured by principal components 1 and 2. PCA obviates the customary focus on specific peaks or regions of images. Applying it directly to a series of complex data will easily delineate binding isotherms, equilibrium shifts, and time courses of reactions or fluctuations

Remote Exosites of the Catalytic Domain of Matrix Metalloproteinase-12 Enhance Elastin Degradation

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? Nuclear magnetic resonance suggested soluble elastin covers surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II–III loop and β-strand I near the back of the catalytic domain. The high degree of exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, the combination of one lesion each at exosites 1 and 2 and the active site decreased the catalytic competence toward soluble elastin by 13–18-fold to the level of MMP-3, homologue and poor elastase. Double-mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) showed they had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 the <i>k</i>_cat for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced the <i>k</i>_cat for soluble elastin. In elastin degradation, exosites 1 and 2 contributed in a manner independent of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils