Alireza Mashaghi

Surface Functionalization of Single Superparamagnetic Iron Oxide Nanoparticles for Targeted Magnetic Resonance Imaging

Magnetic resonance imaging (MRI), a non-invasive, non-radiative technique, is thought to lead to cellular or even molecular resolution if optimized targeted MR contrast agents are introduced. This would allow diagnosing progressive diseases in early stages. Here, it is shown that the high binding affinity of poly(ethylene glycol)-gallol (PEG-gallol) allows freeze drying and re-dispersion of 9 +/- 2-nm iron oxide cores individually stabilized with approximately 9-nm-thick stealth coatings, yielding particle stability for at least 20 months. Particle size, stability, and magnetic properties of PEGylated particles are compared to Feridex, a commercially available untargeted negative MR contrast agent. Biotin-PEG(3400)-gallol/methoxy-PEG(550)-gallol stabilized nanoparticles are further functionalized with biotinylated human anti-VCAM-1 antibodies using the biotin-neutravidin linkage. Binding kinetics and excellent specificity of these nanoparticles are demonstrated using quartz crystal microbalance with dissipation monitoring (QCM-D). These MR contrast agents can be functionalized with any biotinylated ligand at controlled ligand surface density, rendering them a versatile research tool.

Optical Anisotropy of Supported Lipid Structures Probed by Waveguide Spectroscopy and Its Application to Study of Supported Lipid Bilayer Formation Kinetics

Supramolecular conformation and molecular orientation was monitored during supported lipid bilayer (SLB) formation using dual polarization interferometry (DPI). DPI was shown to enable real time sensitive determination of birefringence of the lipid bilayer together with thickness or refractive index (with the other a fixed value). This approach removes differences in mass loading due to anisotropy, so the mass becomes solely a function of the lipid d n/d c value. DPI measurements show highly reproducible qualitative and quantitative results for adsorption of liposomes of different lipid compositions and in buffers with or without CaCl 2. The packing of solvent-free self-assembled SLBs is shown to differ from other preparation methods. Birefringence analysis accompanied by mass and thickness measurements shows characteristic features of vesicle adsorption and SLB formation kinetics previously not demonstrated by evanescent optical techniques, including indications of percolation-type rupture of clusters of liposomes on the surface and correlated adsorption kinetics induced by liposome charge repulsion. Our study demonstrates that understanding of mechanistic details for an adsorption process for which conformational changes and ordering occur can be elucidated using DPI and greatly enhanced by modeling of optical birefringence. The data is in some respects more detailed than what can be obtained with conventional biosensing techniques like surface plasmon resonance and complementary to methods such as the quartz crystal microbalance.

Calcium modulates force sensing by the von Willebrand factor A2 domain.

von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing.

Reshaping of the conformational search of a protein by the chaperone trigger factor

Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts, as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself—by reshaping the underlying folding landscape along the folding trajectory—direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.

Alternative modes of client binding enable functional plasticity of Hsp70

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Hydration strongly affects the molecular and electronic structure of membrane phospholipids

We investigate the structure and electronic properties of phosphatidylcholine (PC) under different degrees of hydration at the single-molecule and monolayer type level by linear scaling ab initio calculations. Upon hydration, the phospholipid undergoes drastic long-range conformational rearrangements which lead to a sickle-like ground-state shape. The structural unit of the tilted gel-phase PC appears to be a water-bridged PC dimer. We find that hydration dramatically alters the surface potential, dipole and quadrupole moments of the lipids and consequently guides the interactions of the lipids with other molecules and the communication between cells.

Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

Chaperone Action at the Single-Molecule Level

Alireza Mashaghi,*,† Günter Kramer,‡ Don C. Lamb, Matthias P. Mayer,‡ and Sander J. Tans*,† †AMOLF Institute, Science Park 104, 1098 XG Amsterdam, The Netherlands ‡Zentrum für Molekulare Biologie der Universitaẗ Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Physical Chemistry, Department of Chemistry, Munich Center for Integrated Protein Science (CiPSM) and Center for Nanoscience, Ludwig-Maximilians-Universitaẗ München, Butenandtstrasse 5-13, Gerhard-Ertl-Building, 81377 Munich, Germany

Neuropeptide substance P and the immune response

Substance P is a peptide mainly secreted by neurons and is involved in many biological processes, including nociception and inflammation. Animal models have provided insights into the biology of this peptide and offered compelling evidence for the importance of substance P in cell-to-cell communication by either paracrine or endocrine signaling. Substance P mediates interactions between neurons and immune cells, with nerve-derived substance P modulating immune cell proliferation rates and cytokine production. Intriguingly, some immune cells have also been found to secrete substance P, which hints at an integral role of substance P in the immune response. These communications play important functional roles in immunity including mobilization, proliferation and modulation of the activity of immune cells. This review summarizes current knowledge of substance P and its receptors, as well as its physiological and pathological roles. We focus on recent developments in the immunobiology of substance P and discuss the clinical implications of its ability to modulate the immune response.

Label-free characterization of biomembranes: From structure to dynamics

We review recent progress in the study of the structure and dynamics of phospholipid membranes and associated proteins, using novel label-free analytical tools. We describe these techniques and illustrate them with examples highlighting current capabilities and limitations. Recent advances in applying such techniques to biological and model membranes for biophysical studies and biosensing applications are presented, and future prospects are discussed.