Sang-Ho Park

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure–activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an α‐helical structure in trifluoroethanol (TFE)/H2O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H2O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic α‐helix spanning residues 3–18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic α‐helix that potentially disrupts membrane integrity, which would result in cell death.

Role of proline, cysteine and a disulphide bridge in the structure and activity of the anti-microbial peptide gaegurin 5.

Gaegurin 5 (GGN5) is a cationic 24-residue anti-microbial peptide isolated from the skin of a Korean frog, Rana rugosa. It contains a central proline residue and an intra-residue disulphide bridge in its C-terminus, which are common to the anti-microbial peptides found in Ranidae. We determined the solution structure of GGN5 bound to SDS micelles for the first time and investigated the role of proline, cysteine and a disulphide bridge on the structure and activity of GGN5. GGN5 adopts an amphipathic alpha-helical structure spanning residues 3-20 kinked around Pro-14, which allows the hydrophobic residues to reside in the concave helical region, and a disulphide-bridged loop-like conformation in its C-terminus. By replacement of proline with alanine (PAGGN5), a straight and rigid helix was formed in the central region and was more stable than the kinked helix. Reduction of a disulphide bridge in the C-terminus (GGN5SH) maintained the loosely ordered loop-like conformation, while the replacement of two cysteines with serines (CSGGN5) caused the C-terminal conformation to be completely disordered. The magnitude of anti-microbial activity of the peptides was closely related to their helical stability in the order PAGGN5>GGN5>GGN5SH>CSGGN5, suggesting that the helical stability of the peptides is important for anti-microbial activity. On the other hand, the significant increase of haemolytic activity of PAGGN5 implies that a helical kink of GGN5 could be involved in the selectivity of target cells. The location of GGN5 and PAGGN5, analysed using paramagnetic probes, was mainly at the surface of SDS micelles, although the location of the N-terminal region was slightly different between them.

Stoichiometry and Structural Effect of the Cyclic Nucleotide Binding to Cyclic AMP Receptor Protein

Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes. Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous. In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP. A series of1H-15N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed. The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents. Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain. In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.

Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I

The conformational preferences of AnxIN26, a peptide corresponding to residues 2–26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxIN26 adopts a mainly α‐helical conformation in membrane‐mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxIN26 showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp‐12 side‐chain, which was quite different between the two. The N‐terminal region of the AnxIN26 helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N‐terminal tail domain of human annexin I interacts with biological membranes in a partially calcium‐dependent manner.

Letter to the Editor: Backbone NMR assignments of the metal-free UreE from Bacillus pasteurii

Urease is a nickel-containing enzyme that catalyzes the hydrolysis of urea. Increased pH arising from this reaction is critical to the virulence of several human and animal pathogens (Mobley and Hausinger, 1989). UreE, a urease accessory protein, transports nickel ions into the urease active site (Colpas and Hausinger, 2000). The crystal structure of the UreE from Bacillus pasteurii (BpUreE) has been recently solved as a zincbound form, where the C-terminal residues containing the conserved histidines were not visible (Remaut et al., 2001). This crystal structure showed two forms of tetrameric states sharing a zinc ion in the dimerdimer interface. However, it has been revealed that the tetrameric state of BpUreE could be formed only with a highly concentrated sample in the presence of metal ions, and the physiologically relevant unit of BpUreE in solution is a dimer (Ciurli et al., 2002). The BpUreE dimer has a molecular mass of 34.8 kDa, and consists of two identical subunits, each 147 amino acids long. Recently, it has been revealed that the nickel binding increases the stability of BpUreE, probably by altering the conformation or the dynamic property of the protein (Lee et al., 2002). However, the detailed picture of the nickel-binding effect remains obscure at the structural level, since the structure of both the metal-free and the nickel-bound BpUreE is not available. In this report, we describe the complete backbone NMR assignments of the metal-free BpUreE dimer. The results would provide fundamental information for further

Structural studies on the antimicrobial peptide Brevinin 1E by spectroscopic methods

Skin extracts of frogs are a rich source of pharmacologically active peptides such as caeruleins, tachykinins, bradykinins, thyrotropin-releasing hormone, bombesin-like and opioid peptides. A large variety of antimicrobial peptides has been isolated from Rana species. These peptides, grouped in several families on the basis of differing length and distinct activity, were found to have one structural motif in common: an intramolecular disulfide bridge located at the C-terminal end, forming a seven-member ring, which was designated ‘Rana box’. Brevinin 1E is a 24-residue antimicrobial peptide isolated from the skin of a frog, Rana brevipoda. This peptide shows a broad range of antimicrobial activity against prokaryotic cells but shows very much hemolytic activity against human red blood cells. The solution structure of Brevinin 1E was studied by using CD (circular dichroism) and NMR (nuclear magnetic resonance) spectroscopy. CD investigation revealed that Brevinin 1E adopts random structure in aqueous solution but adopts mainly α-helical structure in TFE/water (6 : 4, v/v) solution. The three-dimensional structure of Brevinin 1E was determined in 60% TFE/water solution using homonuclear NMR spectroscopy. This peptide showed mainly an α-helical structure with amphipathic property. Its three-dimensional structure is similar to those of other peptides such as magainin, nigrocin and ranalexin. Therefore, Brevinin 1E can be classified into the family of antimicrobial peptides containing a single linear α-helix that interact with target microbial membrane, leading to cell death through disruption of membrane integrity.

Structure-Activity Relationship of Antimicrobial Peptide, Gaegurin 4, Isolated from Korean Frog

Gaegurin 4 is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. It shows a broad range of activity against prokaryotic cells, but very little hemolytic activity against human red blood cells [1]. Using circular dichroism and NMR spectroscopy, we have determined the solution structure of gaegurin 4 [2]. In the present study, we determined the structure of this peptide bound to sodium dodecyl sulfate micelles and investigated the structure-activity relationship of this peptide by comparing the structure and physicochemical properties with other known antimicrobial peptides.