Karin Nienhaus

Engineered nanoparticles interacting with cells: size matters

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.

Structural Characterization of Irisfp, an Optical Highlighter Undergoing Multiple Photo-Induced Transformations.

Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis–trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated π-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis–trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.

The structure of murine neuroglobin: Novel pathways for ligand migration and binding

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 Å reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 Å3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high‐resolution structure, further structure‐function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia. Proteins 2004.

mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.

Ligand binding and protein dynamics in neuroglobin.

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O2 and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3–353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275–353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 μs); (ii) extremely fast bimolecular exogenous ligand binding (10 μs < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 μs < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.

Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications

Abstract EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30°C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37°C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy

Summary Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter) carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.

Ligand dynamics in a protein internal cavity.

We have studied the temperature dependence of the IR stretch bands of carbon monoxide (CO) in the Xe 4 internal cavity of myoglobin mutant L29W-S108L at cryogenic temperatures. Pronounced changes of band areas and positions were analyzed quantitatively by using a simple dynamic model in which CO rotation in the cavity is constrained by a static potential. The librational dynamics of the CO causes a decrease of the total spectral area. A strong local electric field splits the CO stretch absorption into a doublet, indicating that CO can assume opposite orientations in the cavity. With increasing temperature, the two peaks approach each other, because the average angle of the CO with respect to the electric field increases. A combined classical and quantum-mechanical analysis precisely reproduces the observed temperature dependencies of both spectral area and peak shifts. It yields the height of the energy barrier between the two wells associated with opposite CO orientations, V0 ≈ 2 kJ/mol, and the frequency of oscillation within a well, ω ≈ 25 cm-1. The electric field in the protein cavity was estimated as 10 MV/cm.

Structural Basis of Enhanced Photoconversion Yield in Green Fluorescent Protein-Like Protein Dendra2.

Dendra2 is an engineered, monomeric GFP-like protein that belongs to a subclass of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. This photoinduced process occurs only in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized pi-electron system. We have measured the X-ray structure of the green species of Dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. The structure, which is very similar to those reported for the closely related proteins EosFP and Kaede, revealed a local structural change involving mainly Arg66 and a water molecule W4, which are part of a charged and hydrogen-bonded cluster of amino acids and water molecules next to the chromophore. Unlike in EosFP and Kaede, Arg66 of Dendra2 does not contribute to negative charge stabilization on the imidazolinone ring by hydrogen bonding to the imidazolinone carbonyl. This structural change may explain the blue shift of the absorption and emission bands, as well as the markedly higher pKs of the hydroxyphenyl moiety of the chromophore, which were determined as 7.1 and 7.5 for the green and red species, respectively. The action spectrum of photoconversion coincides with the absorption band of the neutral species. Consequently, its 20-fold enhancement in Dendra2 at physiological pH accounts for the higher photoconversion yield of this protein as compared to EosFP.

Structural dynamics of myoglobin: Ligand migration among protein cavities studied by fourier transform infrared/temperature derivative spectroscopy

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A0 and A3, which can be distinguished by their different CO bands near 1965 and 1933 cm−1. They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C′, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C′′) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ∼180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C′, C′′, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C′) is severely restricted by introduction of the bulky Phe side chain at position 107.