Production, purification, and quality assessment of borrelial proteins CspZ from Borrelia burgdorferi and FhbA from Borrelia hermsii.

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH15-20 and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1.

Inviting C5-Trifluoromethylated Pseudoprolines into Collagen Mimetic Peptides.

Numerous collagen mimetic peptides (CMPs) have been engineered using proline derivatives substituted at their C(3) and/or C(4) position in order to stabilize or functionalize collagen triple-helix mimics. However, no example has been reported so far with C(5) substitutions. Here, we introduce a fluorinated CMP incorporating trifluoromethyl groups at the C(5) position of pseudoproline residues. In tripeptide models, our CD, NMR, and molecular dynamics (MD) studies have shown that, when properly arranged, these residues meet the structural requirements for a triple-helix assembly. Two host-guest CMPs were synthesized and analyzed by CD spectroscopy. The NMR analysis in solution of the most stable confirmed the presence of structured homotrimers that we interpret as triple helices. MD calculations showed that the triple-helix model remained stable throughout the simulation with all six trifluoromethyl groups pointing outward from the triple helix. Pseudoprolines substituted at the C(5) positions appeared as valuable tools for the design of new fluorinated collagen mimetic peptides.

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone.

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin.

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth.

The peptide KLA (acetyl-(KLAKLAK)2-NH2), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen), by a disulfide bridge. Remarkably, the conjugate KLA-Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA-Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA-Pen. The interaction with membrane models of KLA-Pen, KLA and penetratin were studied using dynamic light scattering, calorimetry, plasmon resonance, circular dichroism and ATR-FTIR to unveil the mode of action of the conjugate. To understand the selectivity of the conjugate towards tumor cell lines and its action on mitochondria, lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. KLA-Pen may exert its deleterious and selective action on cancer cells by the formation of pores with an oblique membrane orientation and establishment of important hydrophobic interactions. These results suggest that KLA-Pen could be a lead compound for the design of cancer therapeutics.

NMR structure of a viral peptide inserted in artificial membranes: a view on the early steps of the birnavirus entry process.

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5-8 and 2-4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by (1)H NMR studies provide structural insights of the pep46 domain interaction. In CDCl(3)/CD(3)OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.

Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies.

UNLABELLED: Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH(2). One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-009-0031-9) contains supplementary material, which is available to authorized users.

The role of membranes in the organization of HIV-1 Gag p6 and Vpr: p6 shows high affinity for membrane bilayers which substantially increases the interaction between p6 and Vpr.

The molecular mechanism by which HIV-1 Gag proteins are targeted and transported to the plasma membrane after ribosomal synthesis is unknown. In this work, we investigated the potential interaction of p6 and Vpr with model membranes and have determined their binding constants. Plasmon waveguide resonance (PWR) experiments showed that p6 strongly interacts with membranes (K(d) approximately 40 nM), which may help explaining in part why Gag is targeted to and assembles into membranes by coating itself with lipids. Moreover, a substantial increased affinity of Vpr for p6 was observed while in a membrane environment. In order to further investigate the molecular properties behind the high affinity to model membranes, molecular dynamics simulations were carried out for p6 with a dodecylphosphocholine (DPC) micelle. The results indicate an integration route model for Vpr into virions and may help explain why previous reports failed to detect p6 in virion core preparations.

Structural studies of HIV-1 Gag p6ct and its interaction with Vpr determined by solution nuclear magnetic resonance.

The ability of human immunodeficiency virus type 1 (HIV-1) to egress from human cells by budding with the cell membrane remains a complex phenomenon of unclear steps. HIV-1 viral protein R (Vpr) incorporation in sorting virions relies greatly on the interaction with the group-specific antigen (Gag) C-terminal region, which encompasses protein p6. The complete role of p6 is still undetermined; however, it is thought that p6 interacts with protein core elements from the endosomal sorting complex ESCRT-1, known to sort ubiquitinated cargo into multivesicular bodies (MVB). The three-dimensional structure of the p6 C-terminus (p6ct) comprising amino acids 32-52, determined in this study using NMR methods, includes the region thought to interact with Vpr, i.e., the LXXLF sequence. Here we present new results indicating that the region which interacts with Vpr is the ELY(36) sequence, in the same region where mutational studies revealed that replacing Y36 with a phenylalanine would increase the infectivity of virions by 300-fold. The interaction of Vpr with an egg PC bilayer in the presence of p6ct measured by plasmon waveguide resonance (PWR) is approximately 0.8 microM, approximately 100 times stronger in the absence of p6ct. Our results suggests an interaction based on an ELYP(37) sequence bearing similarities with recently published results, which elegantly demonstrated that the HIV-1 Gag LYPx(n)LxxL motif interacts with Alix 364-702. Moreover, we performed a 60 ns molecular dynamics (MD) simulation of p6ct in DPC micelles. The MD results, supported by differential scanning calorimetry measurements in DMPC, show that p6ct adsorbs onto the DPC micelle surface by adopting a rather stable alpha-helix. Our results provide insights regarding the HIV-1 virion sorting mechanism, specifically concerning the interaction between p6 and Vpr. We also suggest that Gag p6 may adsorb onto the surface of membranes during the sorting process, a property so far only attributed to the N-terminal portion of Gag matrix (MA), which is myristylated. The implications of such a novel event provide an alternative direction toward understanding the assembly and escape mechanisms of virions, which have been undetected so far.