Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) are associated with dysregulation and overactivation of the complement alternative pathway. Typically, gene analysis for aHUS and C3G is undertaken in small patient numbers, yet it is unclear which genes most frequently predispose to aHUS or C3G. Accordingly, we performed a six-center analysis of 610 rare genetic variants in 13 mostly complement genes (, ,, ,, ,, ,, ,, , and) from >3500 patients with aHUS and C3G. We report 371 novel rare variants (RVs) for aHUS and 82 for C3G. Our new interactive Database of Complement Gene Variants was used to extract allele frequency data for these 13 genes using the Exome Aggregation Consortium server as the reference genome. For aHUS, significantly more protein-altering rare variation was found in five genes ,, ,, and than in the Exome Aggregation Consortium (allele frequency< 0.01%), thus correlating these with aHUS. For C3G, an association was only found for RVs in and the N-terminal C3b-binding or C-terminal nonsurface-associated regions of In conclusion, the RV analyses showed nonrandom distributions over the affected proteins, and different distributions were observed between aHUS and C3G that clarify their phenotypes.

Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion.