Probing structural elements in RNA using engineered disulfide cross-links.

Cross-Linking Electrochemical Mass Spectrometry for Probing Protein Three-Dimensional Structures

Chemical cross-linking combined with mass spectrometry (MS) is powerful to provide protein three-dimensional structure information but difficulties in identifying cross-linked peptides and elucidating their structures limit its usefulness. To tackle these challenges, this study presents a novel cross-linking MS in conjunction with electrochemistry using disulfide-bond-containing dithiobis[succi...

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages.

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined t...

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4.

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the...

PROBING THE ACTIVE SITE OF RHODANESE WITH DISULFIDE REAGENTS

Noncovalent Cross-links in Context with Other Structural and Functional Elements of Proteins

Proteins are heteropolymers with evolutionary selected native sequences of residues. These native sequences code for unique and stable 3D structures indispensable for biochemical activity and for proteolysis resistance, the latter which guarantees an appropriate lifetime for the protein in the protease rich cellular environment. Cross-links between residues close in space but far in the primary...