David Paramelle

Chemical cross‐linkers for protein structure studies by mass spectrometry

The cross‐linking approach combined with MS for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low‐abundance cross‐linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over‐represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, because of the outstanding technical developments in MS. This improvement greatly simplified the identification of cross‐linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross‐linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross‐links, or the interpretation of MS data. These chemical features are reviewed and commented on in the light of the more recent strategies.

A new generation of cross-linkers for selective detection by MALDI MS

We designed a new cross‐linker bearing a CHCA moiety. The use of the CHCA‐tagged cross‐linker JMV 3378 in conjunction with a neutral MALDI matrix α‐cyano‐4‐hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI‐TOF MS of cross‐link containing peptides. Discrimination between modified and non‐modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the α‐cyano‐4‐hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo‐myoglobine as model proteins.

Features of Thiolated Ligands Promoting Resistance to Ligand Exchange in Self-Assembled Monolayers on Gold Nanoparticles

An important feature necessary for biological stability of gold nanoparticles is resistance to ligand exchange. Here, we design and synthesize self-assembled monolayers of mixtures of small ligands on gold nanoparticles promoting high resistance to ligand exchange. We use as ligands short thiolated peptidols, e.g. H-CVVVT-ol, and ethylene glycol terminated alkane thiols (HS-C11-EG4). We present a straightforward method to evaluate the relative stability of each ligand shell against ligand exchange with small thiolated molecules. The results show that a ligand with a ‘thin’ stem, such as HS-C11-EG4, is an important feature to build a highly packed self-assembled monolayer and provide high resistance to ligand exchange. The greatest resistance to ligand exchange was found for the mixed ligand shells of the pentapeptidols H-CAVLT-ol or H-CAVYT-ol and the ligand HS-C11-EG4 at 30:70 (mole/mole). Mixtures of ligands of very different diameters, such as the peptidol H-CFFFY-ol and the ligand HS-C11-EG4, provide only a slightly lower stability against ligand exchange. These ligand shells are thus likely to be suitable for long-term use in biological environments. The method developed here provides a rapid screening tool to identify nanoparticles likely to be suitable for use in biological and biomedical applications.

Designing Non‐Native Iron‐Binding Site on a Protein Cage for Biological Synthesis of Nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages.

A Straightforward Approach for Cellular‐Uptake Quantification

Cell-penetrating compounds are able to cross biological membranes and deliver bioactive cargo into cell compartments (cytoplasm, nucleus). Different methods of detection have been employed to study their cellular uptake. Fluorescent dyes and radioactive labels are commonly used. However, direct quantification of internalized compounds is a lot more difficult, and different studies have led to different results. The pioneering study of Burlina et al. constituted a real breakthrough in proposing a highly reproducible quantification method based on MALDI-TOF MS to measure the concentration of the internalized peptides. After cell lysis, this method requires the capture of the biotin-labeled cellpenetrating peptides (CPPs). This step is particularly critical for the accuracy of the quantification. Indeed, the lysate may contain molecules that may hamper the CPP capture by streptavidin-coated magnetic beads. However, the attractiveness of such an MS-based methodology for accurate CPP quantification from complex biological media could be greatly enhanced by avoiding affinity labeling and subsequent purification. We report herein a sensitive general method for the quantification of internalized compounds into cells by MALDI-TOF mass spectrometry that combines existing analytical tools for highly sensitive peptide detection and very accurate protein/peptide quantitation. We previously reported an original approach in which peptides derivatized by a-cyano-4-hydroxycinnamic acid (HCCA) were readily identified by selective enhancement and discrimination of the MALDI MS signals in a neutral matrix, such as a-cyano-4hydroxycinnamic methyl ester (HCCE). This combination (HCCA tag and HCCE matrix) enabled us to discriminate signals induced by peptides of interest that were present in low concentrations from those of unlabeled more abundant peptides. Reliable accurate measurement of protein expression was demonstrated in quantitative proteomics by Oda et al., who used a stable-isotope-labeling MS-based strategy. We therefore decided to prepare a heavy (D4) analogue of the UV-light-absorbing label HCCA for the quantification of CPP cellular uptake. We synthesized CPPs coupled with light (D0) or heavy (D4) HCCA through an aminohexanoic acid (Ahx) spacer (Figure 1A). Ahx is commonly used as a spacer between cargo and CPPs to prepare N-terminally tagged conjugates. 6] The ability of these compounds to penetrate cells was readily determined by comparison of the MS signals induced by tagged compounds with those of the overrepresented untagged materials. Thus, no separation procedure was required. The material that penetrated cells was quantified by comparison of the signals due to the light tag with the corresponding signals corresponding to deuterated heavy HCCA. The methodology (described in Figure 1B) was validated by using four different compounds: the two widely used CPPs penetratin and nonaarginine (Arg)9, [7, 8] the benzothiazepine-derived oligomer (DBT)4, which we previously identified as a potent cell-penetrating nonpeptide (CPNP), and a tripeptide (FAK) as a negative control (Table 1). All compounds were prepared by microwaveassisted solid-phase synthesis. Figure 1A summarizes the synthetic and analytic workflows. The key step of the synthesis was a Knoevenagel condensation with commercially available deuterated p-hydroxybenzaldehyde. Before performing internalization experiments, we checked that HCCA-tagged peptides could be detected in a crude cell lysate by MALDI-TOF MS up to a 10 m concentration (see Figure S1 in the Supporting information), which corresponds to the possible concentration of internalized compound in the sample after cell lysis. To highlight the HCCE/HCCA matrix-discrimination effect, N-terminal acetylated peptides were prepared and mixed at different concentrations along with HCCA-tagged peptides in a crude cell lysate. Equimolar mixtures of HCCA-CPPs and Ac-CCPs diluted in water/acetonitrile were mixed in a cell lysate to afford a 5 10 6 to 5 10 m concentration of each peptide species. Samples were prepared either in an HCCA matrix or in a neutral HCCE matrix to assess the discrimination effect (see Figure S1 in the Supporting Information). The MALDI-TOF spectra were quite clean, and very few signals were observed for the cell lysate or the buffer. HCCAtagged peptides were still readily detected at 5 10 m in the HCCA matrix and 5 10 m in the HCCE matrix. Ac-CPPs were not detected at a concentration of 5 10 m. These [*] Dr. D. Paramelle, Dr. G. Subra, L. L. Vezenkov, C. Andr , Prof. C. Enjalbal, Dr. M. Calm s, Prof. J. Martinez, Dr. M. Amblard Institut des Biomol cules Max Mousseron (IBMM) UMR5247 CNRS, Universit s Montpellier 1 et 2 15 avenue Charles Flahault, 34000 Montpellier (France) Fax: (+ 33)4-6754-8654 E-mail: gilles.subra@univ-montp1.fr muriel.amblard@univ-montp1.fr Homepage: http://www.ibmm.univ-montp1.fr/

Synthesis of Silver Nanoparticles with Monovalently Functionalized Self-Assembled Monolayers

The importance of having nanoparticles that are soluble, stable, and that have no non-specific binding is often overlooked, but essential for their use in biology. This is particularly prominent with silver nanoparticles that are susceptible to the effects of aggregation and metal-surface reactivity. Here we use a combination of several small peptidols and short alkanethiol ethylene glycol ligands to develop a ligand shell that is reasonably resistant to ligand exchange and non-specific binding to groups common in biological molecules. The stability of the nanoparticles is not affected by the inclusion of a functional ligand, which is done in the same preparative step. The stoichiometry of the nanoparticles is controlled, such that monofunctional silver nanoparticles can be obtained. Two different sets of nanoparticles, functionalized with either Tris-nitrilotriacetic acid or a hexa-histidine peptide sequence, readily form dimers/oligomers, depending on their stoichiometry of functionalization.

Targeting Cell Membrane Lipid Rafts by Stoichiometric Functionalization of Gold Nanoparticles with a Sphingolipid-Binding Domain Peptide

A non-membrane protein-based nanoparticle agent for the tracking of lipid rafts on live cells is produced by stoichiometric functionalization of gold nanoparticles with a previously characterized sphingolipid- and cell membrane microdomain-binding domain peptide (SBD). The SBD peptide is inserted in a self-assembled monolayer of peptidol and alkane thiol ethylene glycol, on gold nanoparticles surface. The stoichiometric functionalization of nanoparticles with the SBD peptide, essential for single molecule tracking, is achieved by means of non-affinity nanoparticle purification. The SBD-nanoparticles have remarkable long-term resistance to electrolyte-induced aggregation and ligand-exchange and have no detectable non-specific binding to live cells. Binding and diffusion of SBD-nanoparticles bound to the membrane of live cells is measured by real-time photothermal microscopy and shows the dynamics of sphingolipid-enriched microdomains on cells membrane, with evidence for clustering, splitting, and diffusion over time of the SBD-nanoparticle labeled membrane domains. The monofunctionalized SBD-nanoparticle is a promising targeting agent for the tracking of lipid rafts independently of their protein composition and the labelling requires no prior modification of the cells. This approach has potential for further functionalization of the particles to manipulate the organization of, or targeting to microdomains that control signaling events and thereby lead to novel diagnostics and therapeutics.

Computational and Experimental Investigation of the Structure of Peptide Monolayers on Gold Nanoparticles

The self-assembly and self-organization of small molecules on the surface of nanoparticles constitute a potential route toward the preparation of advanced proteinlike nanosystems. However, their structural characterization, critical to the design of bionanomaterials with well-defined biophysical and biochemical properties, remains highly challenging. Here, a computational model for peptide-capped gold nanoparticles (GNPs) is developed using experimentally characterized Cys-Ala-Leu-Asn-Asn (CALNN)- and Cys-Phe-Gly-Ala-Ile-Leu-Ser-Ser (CFGAILSS)-capped GNPs as a benchmark. The structure of CALNN and CFGAILSS monolayers is investigated using both structural biology techniques and molecular dynamics simulations. The calculations reproduce the experimentally observed dependence of the monolayer secondary structure on the peptide capping density and on the nanoparticle size, thus giving us confidence in the model. Furthermore, the computational results reveal a number of new features of peptide-capped monolayers, including the importance of sulfur movement for the formation of secondary structure motifs, the presence of water close to the gold surface even in tightly packed peptide monolayers, and the existence of extended 2D parallel β-sheet domains in CFGAILSS monolayers. The model developed here provides a predictive tool that may assist in the design of further bionanomaterials.

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies.

A wide range of chemical reagents are available to study the protein–protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X‐ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid‐supported cleavable monofunctional reagents and cross‐linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid‐Phase Cross‐Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over‐represented unmodified materials.

Experimental and Computational Investigation of the Structure of Peptide Monolayers on Gold Nanoparticles

The self-assembly and self-organization of small molecules at the surface of nanoparticles constitute a potential route towards the preparation of advanced protein-like nanosystems. However, their structural characterization, critical to the design of bio-nanomaterials with well-defined biophysical and biochemical properties, remains highly challenging. Here, a computational model for peptide-capped gold nanoparticles is developed using experimentally characterized CALNN-and CFGAILSS-capped gold nanoparticles as a benchmark. The structure of CALNN and CFGAILSS monolayers is investigated by both structural biology techniques and molecular dynamics simulations. The calculations reproduce the experimentally observed dependence of the monolayer secondary structure on peptide capping density and on nanoparticle size, thus giving us confidence in the model. Furthermore, the computational results reveal a number of new features of peptide-capped monolayers, including the importance of sulfur movement for the formation of secondary structure motifs, the presence of water close to the gold surface even in tightly packed peptide monolayers, and the existence of extended 2D parallel β-sheet domains in CFGAILSS monolayers. The model developed here provides a predictive tool that may assist in the design of further bio-nanomaterials.