Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules.

BP100 is a multifunctional membrane-active peptide of only 11 amino acids, with a high antimicrobial activity, an efficient cell-penetrating ability, and low hemolytic side-effects. It forms an amphiphilic α-helix, similar to other antimicrobial peptides like magainin. However, BP100 is very short and thus unlikely to form membrane-spanning pores as proposed for longer peptides as a mechanism of action. We thus studied the conformation, membrane alignment and dynamical behavior of BP100 in lipid bilayers (DMPC/DMPG), using oriented circular dichroism (OCD) and solid-state (19)F and (15)N NMR. According to OCD and (15)N NMR, the BP100 helix is oriented roughly parallel to the membrane surface, but these methods yield no information on the azimuthal alignment angle or the dynamics of the molecule. To address these questions, a systematic (19)F NMR analysis was performed, which was not straightforward for this short peptide. Only a limited number of positions could be (19)F-labeled, all of which are located on one face of the helix, which was found to lead to artifacts in the data analysis. It was nevertheless possible to reconcile the (19)F NMR data with the OCD and (15)N NMR data by using an advanced dynamical model, in which peptide mobility is described by fluctuating tilt and azimuthal angles with Gaussian distributions. (19)F NMR thus confirmed the regular α-helical conformation of BP100, revealed its azimuthal angle, and described its high mobility in the membrane. Furthermore, the very sensitive (19)F NMR experiments showed that the alignment of BP100 does not vary with peptide concentration over a peptide-to-lipid molar ratio from 1:10 to 1:3000.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. (19)F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively (19)F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. (31)P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, (2)H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

Bacterial mechanosensitive channels protect cells from structural damage during hypoosmotic shock. MscS, MscL and MscK are the most abundant channels in E. coli and arguably the most important ones in osmoprotection. By combining physiological assays with quantitative photo-activated localization microscopy (qPALM), we find an almost linear relationship between channel abundance and cell survival. A minimum of 100 MscL (or MscS) channels is needed for protection when a single type of channel is expressed. Under native-like conditions MscL, MscS as well as MscK distribute homogeneously over the cytoplasmic membrane and the lateral diffusion of the channels is in accordance with their relative protein mass. However, we observe cluster formation and a reduced mobility of MscL when the majority of the subunits of the pentameric channel contain the fluorescent mEos3.2 protein. These data provide new insights into the quantitative biology of mechanosensitive channels and emphasizes the need for care in analysing protein complexes even when the fluorescent tag has been optimized for monomeric behaviour.