Selective Enrichment and Identification of Azide-tagged Cross-Linked Peptides Using Chemical Ligation and Mass Spectrometry

Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with sample handling. LC MSn analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry

Large vector spaces of block-symmetric strong linearizations of matrix polynomials

Given a matrix polynomial P(lambda) = Sigma(k)(i=0) lambda(i) A(i) of degree k, where A(i) are n x n matrices with entries in a field F, the development of linearizations of P(lambda) that preserve whatever structure P(lambda) might posses has been a very active area of research in the last decade. Most of the structure-preserving linearizations of P(lambda) discovered so far are based on certain modifications of block-symmetric linearizations. The block-symmetric linearizations of P(lambda) available in the literature fall essentially into two classes: linearizations based on the so-called Fiedler pencils with repetition, which form a finite family, and a vector space of dimension k of block-symmetric pencils, called DL(P), such that most of its pencils are linearizations. One drawback of the pencils in DL(P) is that none of them is a linearization when P(lambda) is singular. In this paper we introduce new vector spaces of block,symmetric pencils, most of which are strong linearizations of P(lambda). The dimensions of these spaces are O(n(2)), which, for n >= root k, are much larger than the dimension of DL(P). When k is odd, many of these vector spaces contain linearizations also when P(lambda) is singular. The coefficients of the block-symmetric pencils in these new spaces can be easily constructed as k x k block-matrices whose n x n blocks are of the form 0, +/-alpha I-n, +/-alpha A(i), or arbitrary n x n matrices, where a is an arbitrary nonzero scalar.The research of F. M. Dopico was partially supported by the Ministerio de Economía y Competitividad of Spain through grant MTM-2012-3254

(+)-Hexacyclinol

A sample of the title compound [systematic name: (1aS,2aS,3S,5aS,6aS,7R,7aS,7bS,8R,8aS,10R)-7-hydr­oxy-3-(1-meth­oxy-1-methyl­ethyl)-10-(2-methyl-1-propen­yl)-1a,5a,6a,7,7a,7b,8,8a-octa­hydro-2H-8,2a-(epoxy­methano)phenanthro[2,3-b:6,7-b′]bis­oxirene-2,5(3H)-dione], C23H28O7, was generated by enanti­oselective synthesis. There are three mol­ecules of the compound in the crystallographic asymmetric unit. Hydrogen bonding between alcohol H atoms and keto groups of adjacent mol­ecules appears to stabilize the structure. The compound is enanti­omerically pure but the absolute configuration could not be determined directly in this study. Accordingly, the illustrated configuration was assigned on the basis of the nature of the chiral nonracemic precursor used in the synthesis

Stereodivergent Synthesis of Enantioenriched 4-Hydroxy-2- cyclopentenones

Protected 4-hydroxycyclopentenones (4-HCPs) constitute an important class of intermediates in chemical synthesis. A route to this class of compound has been developed. Key steps include Noyori reduction (which establishes the stereochemistry of the product), ring-closing metathesis, and simple functional group conversions to provide a set of substituted 4-HCPs in either enantiomeric form

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol–channel protein interactions

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself