Giovanna Ghirlanda

Photo-induced hydrogen production in a helical peptide incorporating a [FeFe] hydrogenase active site mimic

There is growing interest in the development of hydrogenase mimics for solar fuel production. Here, we present a bioinspired mimic designed by anchoring a diiron hexacarbonyl cluster to a model helical peptide via an artificial dithiol amino acid. The [FeFe]-peptide complex catalyses photo-induced production of hydrogen in water.

Water oxidation by a nickel-glycine catalyst

The utilization of solar energy requires an efficient means for its storage as chemical energy. In bioinspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are needed to avoid excessive driving potentials. In this paper, we demonstrate the utility of a novel complex utilizing earth-abundant Ni in combination with glycine as an efficient catalyst with a modest overpotential of 0.475 ± 0.005 V at a current density of 1 mA/cm(2) at pH 11. Catalysis requires the presence of the amine moiety with the glycine most likely coordinating the Ni in a 4:1 molar ratio. The production of molecular oxygen at a high potential is verified by measurement of the change in oxygen concentration, yielding a Faradaic efficiency of 60 ± 5%. The catalytic species is most likely a heterogeneous Ni-hydroxide formed by electrochemical oxidation. This Ni species can achieve a current density of 4 mA/cm(2) that persists for at least 10 h. Based upon the observed pH dependence of the current amplitude and oxidation/reduction peaks, the catalytic mechanism is an electron-proton coupled process.

De Novo Design of an Artificial Bis[4Fe-4S] Binding Protein

In nature, protein subunits containing multiple iron-sulfur clusters often mediate the delivery of reducing equivalents from metabolic pathways to the active site of redox proteins. The de novo design of redox active proteins should include the engineering of a conduit for the delivery of electrons to and from the active site, in which multiple redox active centers are arranged in a controlled manner. Here, we describe a designed three-helix protein, DSD-bis[4Fe-4S], that coordinates two iron-sulfur clusters within its hydrophobic core. The design exploits the pseudo two-fold symmetry of the protein scaffold, DSD, which is a homodimeric three-helix bundle. Starting from the sequence of the parent peptide, we mutated eight leucine residues per dimer in the hydrophobic core to cysteine to provide the first coordination sphere for cubane-type iron-sulfur clusters. Incorporation of two clusters per dimer is readily achieved by in situ reconstitution and imparts increased stability to thermal denaturation compared to that of the apo form of the peptide as assessed by circular dichroism-monitored thermal denaturation. The presence of [4Fe-4S] clusters in intact proteins is confirmed by UV-vis spectroscopy, gel filtration, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy. Pulsed electron-electron double-resonance experiments have detected a magnetic dipole interaction between the two clusters ~0.7 MHz, which is consistent with the expected intercluster distance of 29-34 Å. Taken together, our data demonstrate the successful design of an artificial multi-iron-sulfur cluster protein with evidence of cluster-cluster interaction. The design principles implemented here can be extended to the design of multicluster molecular wires.

Design of membrane proteins: toward functional systems

Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.

Multivalent interactions with gp120 are required for the anti-HIV activity of Cyanovirin

Cyanovirin‐N (CV‐N) is a cyanobacterial lectin that binds to specific oligomannoses on the surface of gp120, resulting in nanomolar antiviral activity against HIV. In its monomeric form, CV‐N contains two functional carbohydrate‐binding domains, A and B. When refolded at high concentration, the protein can form a domain‐swapped dimer. To clarify the role of multiple‐binding sites in CV‐N, we previously designed a monomeric mutant, P51G‐m4‐CVN, in which the binding site on domain A was rendered ineffective by four mutations (m4); in addition, a hinge region mutation (P51G) hinders the formation of a domain swapped dimer. The protein bound gp120 with diminished affinity and was completely inactive against HIV. Here, we present two mutants, ΔQ50‐m4‐CVN and S52P‐m4‐CVN, which fold exclusively as domain‐swapped dimers while containing the four mutations that abolish domain A. The dimers contain two intact B domains, thus restoring multivalency. ΔQ50‐m4‐CVN and S52P‐m4‐CVN bind gp120 at low‐nanomolar concentrations and recover in part the antiviral activity of wt CV‐N. These results indicate that the number of carbohydrate binding domains, rather than their identity, is crucial to CV‐N functionality.

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 Å resolution

Cyanovirin (CV‐N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild‐type (wt) protein binds with high affinity to mannose‐rich oligosaccharides on the surface of gp120 through two quasi‐symmetric sites, located in domains A and B. We recently reported on a mutant of CV‐N that contained a single functional mannose‐binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 Å resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV‐N. Here, we present the 1.35 Å resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.

PNA-Peptide Assembly in a 3D DNA Nanocage at Room Temperature

Proteins and peptides fold into dynamic structures that access a broad functional landscape; however, designing artificial polypeptide systems is still a great challenge. Conversely, DNA engineering is now routinely used to build a wide variety of 2D and 3D nanostructures from hybridization based rules, and their functional diversity can be significantly expanded through site specific incorporation of the appropriate guest molecules. Here we demonstrate a new approach to rationally design 3D nucleic acid-amino acid complexes using peptide nucleic acid (PNA) to assemble peptides inside a 3D DNA nanocage. The PNA-peptides were found to bind to the preassembled DNA nanocage in 5-10 min at room temperature, and assembly could be performed in a stepwise fashion. Biophysical characterization of the DNA-PNA-peptide complex was performed using gel electrophoresis as well as steady state and time-resolved fluorescence spectroscopy. Based on these results we have developed a model for the arrangement of the PNA-peptides inside the DNA nanocage. This work demonstrates a flexible new approach to leverage rationally designed nucleic acid (DNA-PNA) nanoscaffolds to guide polypeptide engineering.

De novo design of functional proteins: Toward artificial hydrogenases

Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well-folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog

The post-translational modification of proteins by the covalent attachment of carbohydrates to specific side chains, or glycosylation, is emerging as a crucial process in modulating the function of proteins. In particular, the dynamic processing of the oligosaccharide can correlate with a change in function. For example, a potent macrophage-activating factor, Gc-MAF, is obtained from serum vitamin D binding protein (VDBP) by stepwise processing of the oligosaccharide attached to Thr 420 to the core alpha-GalNAc moiety. In previous work we designed a miniprotein analog of Gc-MAF, MM1, by grafting the glycosylated loop of Gc-MAF on a stable scaffold. GalNAc-MM1 showed native-like activity on macrophages (Bogani 2006, J. Am. Chem. Soc. 128 7142-43). Here, we present data on the thermodynamic stability and conformational dynamics of the mono- and diglycosylated forms. We observed an unusual trend: each glycosylation event destabilized the protein by about 1 kcal/mol. This effect is matched by an increase in the mobility of the glycosylated forms, as evaluated by molecular dynamics simulations. An analysis of the solvent-accessible surface area shows that glycosylation causes the three-helix bundle to adopt conformations in which the hydrophobic residues are more solvent exposed. The number of hydrophobic contacts is also affected. These two factors, which are ultimately explained with a change in occupancy for conformers of specific side chains, may contribute to the observed destabilization.

Simultaneous observation of peptide backbone lipid solvation and α-helical structure by deep-UV resonance Raman spectroscopy.

Despite a variety of methodologies aimed at improving membrane protein structure analysis, information about these proteins in their native membrane environments remains scarce. Currently, no structurally sensitive spectroscopic techniques are capable of co-determining ensemble structural content and localized lipid versus aqueous solvation information. Here, we describe the first deep-UV (lex<210 nm) resonance Raman (dUVRR) spectra of a model a-helical peptide embedded in a membrane-mimetic environment, confirming sensitivity to secondary structure content and revealing sensitivity of dUVRR to the lipid solvation of the peptide backbone. Analyses of membrane protein structural dynamics are hampered by the experimental difficulties associated with elucidating structural changes and correlating those changes to their respective solvation by the nonpolar lipid or surfactant versus the aqueous phases. No kinetically amenable spectroscopic techniques are capable of delineating subtle changes in protein structure while simultaneously reporting on that structure’s solvation without protein modification by deuterium exchange, isotope labeling, mutagenesis or post-translational spin/fluorophore labeling. Glimpses of the dynamics and stabilizing forces involved with protein folding and insertion into membranes have recently been gleaned by UV excited resonance Raman spectroscopy focused on excitation wavelengths specific for aromatic residues (lex>220 nm). Deep-UV (lex< 210 nm) excitation, which has been a valuable tool for analyzing the structure of soluble proteins by accessing the p!p* transition of the peptide backbone vibrational modes and their dynamics, has not been previously explored successfully for this class of hydrophobic proteins. The dUVRR protein spectral response consists of four peptide backbone related amide (Am) responses—I (C=O stretching), II (in phase C-H/N-H stretching/bending), III (out of phase C-H/N-H stretching/bending) and S (coupled C-H/N-H bending; alternately referred to as CaHb). [2a] The combinations of Am mode positions and intensities are strongly correlated to the constraints imparted by particular secondary structures with soluble proteins. Solvent interaction and its extent with the peptide backbone can also influence the Am mode spectral positions in dUVRR and IR and intensities in dUVRR alone. Theoretical calculations with Nmethylacetamide (NMA) in different solvent polarities have revealed that the solvent-dependent Am I intensity differences seen in the dUVRR spectra, but not the IR spectra are derived from the sensitivity of the former technique to the polarizability term of the C=O bond. Herein, we present evidence that a surfactant-solubilized protein region also has altered Am mode intensities, especially in the C=O stretching region. As a model for the common a-helical membrane-embedded protein domain, we have examined the de novo designed ME1 peptide, which contains a single hydrophobic a-helical segment encompassing roughly 75 % of the total peptide backbone. Like its parent protein, it is extremely insoluble in aqueous solvents and only forms stable a-helical homodimers within a micellar environment. The dUVRR spectrum using an excitation source of 197 nm of a dodecyl phosphocholine (DPC)-solubilized ME1 sample contains aromatic side chain-derived modes (1180–1210 and 1580–1620 cm ) arising from the single tyrosine and phenylalanine residues within the peptide sequence (Figure 1). Peptide backbone contributions can also be assigned for the Am I (1658 cm ), II (1546 cm ) and III (1260–1340 cm ) modes and a smaller feature where the Am S (1400 cm ) mode would be expected. The Am III mode’s position, coupled to the limited extent of the Am S contribu-