One-Pot Highly Efficient Synthesis of N-Enrich Graphene Quantum Dots as a Photocatalytic Platform for NAD+/NADP+ Reduction.

An efficient photocatalytic regeneration of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) has been carried out by two-electron reduction and protonation of NAD+ /NADP+ , induced by photons in the visible light region. This functional artificial photosynthetic counterpart of the complete energy-trapping occurring in natural photosystem I (PS I) is achieved with nitrogen-enrich graphene quantum dot (N-EGQD) as the light-harvesting photocatalyst. In buffer aqueous solution, this compound photo catalytically recycles a rhodium hydride complex of the type [Cp*Rh(bpy)H]+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2'-bipyridine) which can mediate hydride transfer processes leading to nucleotide co-factor reduction. Very promising yields of 73.31%/78.45% of NADH/NADPH with the excellent thermal stability of N-EGQD photocatalyst is observed. Thus, in this work, an efficient light-harvesting photocatalyst is synthesized for the regeneration of nicotinamide cofactor that has pharmaceuticals application.

Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate.

Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG-protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.

Cys(i)-Lys(i+3)-Lys(i+4) triad: a general approach for PEG-based stabilization of α-helical proteins.

PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs. Modern chemoselective reactions now enable specific placement of a single PEG at any site on a protein surface. However, few rational structure-based guidelines exist for selecting optimal PEGylation sites. Here, we explore the impact of PEGylation on the conformational stability of α-helices using an α-helical coiled coil as a model system. We find that maleimide-based PEGylation of a solvent-exposed i position Cys can stabilize coiled-coil quaternary structure when Lys residues occupy both the i + 3 and i + 4 positions, due to favorable interactions between the PEG-maleimide and the Lys residues. Applying this Cys(i)-Lys(i+3)-Lys(i+4) triad to a solvent-exposed position within the C-terminal helix of the villin headpiece domain leads to similar PEG-based increases in conformational stability, highlighting the possibility of using the Cys(i)-Lys(i+3)-Lys(i+4) triad as a general strategy for PEG-based stabilization of helical proteins.

Criteria for selecting PEGylation sites on proteins for higher thermodynamic and proteolytic stability.

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

Stabilizing impact of N-glycosylation on the WW domain depends strongly on the Asn-GlcNAc linkage.

N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by -0.85 ± 0.12 kcal mol(-1). Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural α-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.

Impact of site-specific PEGylation on the conformational stability and folding rate of the Pin WW domain depends strongly on PEG oligomer length.

Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.

Cell-selective lysis by novel analogues of melittin against human red blood cells and Escherichia coli.

Melittin is a good model antimicrobial peptide to understand the basis of its lytic activities against bacteria and mammalian cells. Novel analogues of melittin were designed by substituting the leucine residue(s) at the "d" and "a" positions of its previously identified leucine zipper motif. A scrambled peptide having the same composition of melittin with altered leucine zipper sequence was also designed. The analogues of melittin including the scrambled peptide showed a drastic reduction in cytotoxicity though they exhibited comparable bactericidal activities. Only melittin but not its analogues localized strongly onto hRBCs and formed pores of approximately 2.2-3.4 nm. However, melittin and its analogues localized similarly onto Escherichia coli and formed pores of varying sizes as tested onto Bacillus megaterium. The data showed that the substitution of hydrophobic leucine residue(s) by lesser hydrophobic alanine residue(s) in the leucine zipper sequence of melittin disturbed its pore-forming activity and mechanism only in hRBCs but not in the tested bacteria.

Design of nontoxic analogues of cathelicidin-derived bovine antimicrobial peptide BMAP-27: the role of leucine as well as phenylalanine zipper sequences in determining its toxicity.

BMAP-27 is a cathelicidin-derived bovine antimicrobial peptide, which shows moderate cytotoxicity and potent antibacterial activity against a wide variety of microorganisms. Despite a number of studies, very little is known about the amino acid sequences of this peptide that controls its antibacterial and cytotoxic activities. Small stretches of phenylalanine and leucine zipper sequences were identified at the N- and C-termini of the molecule, respectively. To understand the structural and functional roles of these sequence elements, we synthesized and characterized several analogues of BMAP-27 after substituting leucine or phenylalanine residue(s) at a and/or d positions of the leucine and phenylalanine zipper sequences, respectively, with alanine. BMAP-27 analogues exhibited significantly reduced cytotoxicity against the human red blood (hRBC) and murine 3T3 cells as compared to that of the wild-type peptide. Interestingly, BMAP-27 and its analogues exhibited comparable antibacterial activity against the selected Gram-positive and Gram-negative bacteria. Moreover, BMAP-27 and its analogues exhibited similar localization and assembly onto the selected bacteria and induced comparable permeability in these cells. However, only BMAP-27, not its analogues, assembled and bound strongly onto the hRBCs and permeabilized them. The results indicated that not only a leucine zipper but also a phenylalanine zipper sequence plays an important role in maintaining the assembly of BMAP-27 on the mammalian cells examined here and cytotoxic activity against them. To the best of our knowledge, this is the first report of the evaluation of structural and functional roles of a phenylalanine zipper sequence in a naturally occurring antimicrobial peptide.

Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences.

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple 'a' position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.