Genetically Encoded Fluorescent Probe for Detection of Heme-Induced Conformational Changes in Cytochrome c.

Cytochrome c (Cytc) is a key redox protein for energy metabolism and apoptosis in cells. The activation of Cytc is composed of several steps, including its transfer to the mitochondrial membrane, binding to cytochrome c heme lyase (CCHL) and covalent attachment to heme. The spectroscopic methods are often applied to study the structural changes of Cytc.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini.

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes.

Substrate-binding guides individual melibiose permeases MelB to structurally soften and to destabilize cytoplasmic middle-loop C3.

The melibiose permease MelB is a well-studied Na-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium.

Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane.

Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays.

Monitoring the antibiotic darobactin modulating the β-barrel assembly factor BamA.

The β-barrel assembly machinery (BAM) complex is an essential component of Escherichia coli that inserts and folds outer membrane proteins (OMPs). The natural antibiotic compound darobactin inhibits BamA, the central unit of BAM. Here, we employ dynamic single-molecule force spectroscopy (SMFS) to better understand the structure-function relationship of BamA and its inhibition by darobactin.

Insertion and folding pathways of single membrane proteins guided by translocases and insertases.

Biogenesis in prokaryotes and eukaryotes requires the insertion of α-helical proteins into cellular membranes for which they use universally conserved cellular machineries. In bacterial inner membranes, insertion is facilitated by YidC insertase and SecYEG translocon working individually or cooperatively. How insertase and translocon fold a polypeptide into the native protein in the membrane is largely unknown.

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies.

Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from to study outer membrane proteins (Omps) in the native membrane environment.

In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities.

Fluorescent proteins are indispensable for selective, quantitative visualization of localization, dynamics, and interactions of key molecular constituents of live cells. Incorporation of fluorescent proteins into an optical cavity can lead to a significant increase in fluorescence signal levels due to stimulated emission and light amplification in the cavity, forming a laser with biological gain medium. Utilization of lasing emission from fluorescent biological molecules can then greatly enhance the performance of fluorescence-based biosensors benefiting from the high sensitivity of non-linear lasing processes to small perturbations in the cavity and the gain medium.