With the rapid advancement of nanoscience and nanotechnology, detailed knowledge of interactions between engineered nanomaterials and cells, tissues and organisms has become increasingly important, especially in regard to possible hazards to human health. This review intends to give an overview of current research on nano-bio interactions, with a focus on the effects of NP size on their interactions with live cells. We summarize common techniques to characterize NP size, highlight recent work on the impact of NP size on active and passive cellular internalization and intracellular localization. Cytotoxic effects are also discussed.

Neuroglobin (Ngb), a globular heme protein expressed in the brain of vertebrates, binds oxygen reversibly, with an affinity comparable to myoglobin (Mb). Despite low sequence identity, the overall 3D fold of Ngb and Mb is very similar. Unlike in Mb, in Ngb the sixth coordination position of the heme iron is occupied by the distal histidine, in the absence of an exogenous ligand. Endogenous ligation has been proposed as a unique mechanism for affinity regulation and ligand discrimination in heme proteins. This peculiarity might be related to the still-unknown physiological function of Ngb. Here, we present the x-ray structure of CO-bound ferrous murine Ngb at 1.7 Å and a comparison with the 1.5-Å structure of ferric bis-histidine Ngb. We have also used Fourier transform IR spectroscopy of WT and mutant CO-ligated Ngb to examine structural heterogeneity in the active site. Upon CO binding, the distal histidine retains (by and large) its position, whereas the heme group slides deeper into a preformed crevice, thereby reshaping the large cavity (Ϸ290 Å 3 ) connecting the distal and proximal heme sides with the bulk. The heme relocation is accompanied by a significant decrease of structural disorder, especially of the EF loop, which may be the signal whereby Ngb communicates hypoxic conditions. This unexpected structural change unveils a heme-sliding mechanism of affinity control that may be of significance to understanding Ngb's role in the pathophysiology of the brain.protein crystallography ͉ hypoxia signaling ͉ conformational changes ͉ binding N euroglobin (Ngb) is a recently discovered vertebrate heme protein expressed in the brain (1). The 3D structure of ferric Ngb (metNgb) from human and mouse has recently been published (2, 3). The protein displays all key determinants of the canonical 3-over-3 ␣-helical globin fold (4) despite the very low sequence identify with canonical hemoglobins and myoglobins (Mbs). However, in contrast to Mbs, the heme iron in metNgb is hexacoordinated by the distal and proximal histidines (His-64 and His-96) in the absence of an exogenous ligand (2, 3); spectroscopic data show hexacoordination also for ferrous deoxy Ngb (1,5,6). Endogenous ligation at the sixth coordination position has already been reported for nonsymbiotic plant Hbs and bacterial Hbs (7,8). Interestingly, whereas sperm whale Mb (swMb) displays only small matrix cavities (9), metNgb contains a huge internal tunnel (Ϸ290 Å 3 ) that connects the distal and proximal sides of the heme to the bulk (2, 3). Such a large cavity implies a substantial free energy cost estimated at several kcal͞mol (10).Hexacoordination has been proposed as a novel mechanism to regulate ligand affinity in heme proteins (5-8, 11, 12) because the covalent bond between the heme iron and the distal His has to be broken for an exogenous ligand to bind. In Ngb, the latter reaction has been observed in flash photolysis experiments, in which recombination of CO was found to occur in two steps. The first process (on microsecond time scales at [CO] ϭ ...

Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradiation at specific wavelengths. They enable regional optical marking in pulse-chase experiments on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resolution. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chemical reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resolution microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many laboratories, there is still room for further improvement of these important markers for live cell imaging.