Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin.

LFB (FKCRRWQWRMKKLGA-HN2) is a 15-residue linear antimicrobial peptide derived from bovine lactoferricin, which has antimicrobial activity similar to that of the intact 25-residue disulfide-cyclized peptide. Previous alanine-scan studies, in which all of the residues in LFB were individually replaced with Ala, showed that the 2 tryptophan (Trp) residues of LFB were crucial to its antimicrobial activity. When either Trp6 or Trp8 was replaced with Ala (LFBA6 and LFBA8, respectively), these 2 peptides were almost devoid of antimicrobial activity. We determined the structures of LFB, LFBA6, and LFBA8 bound to membrane-mimetic SDS micelles using NMR spectroscopy, and studied their interactions with different phospholipid-model membranes. The membrane interactions of LFB exhibited little correlation with its antimicrobial activity, suggesting that the mechanism of action of LFB involves intracellular targets. However, the much higher antimicrobial activity of LFB compared with LFBA6 and LFBA8 might result, in part, from the formation of energetically favorable cation-pi interactions observed only in LFB. Information about the importance of Arg and Trp cation-pi interactions will provide insight for the future design of potent antimicrobial peptidomimetics.     

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.     

Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.     

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities

The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

Structure of the antimicrobial peptide tritrpticin bound to micelles: a distinct membrane-bound peptide fold

Tritrpticin is a member of the cathelicidin family, a group of diverse antimicrobial peptides found in neutrophil granules. The three Trp and four Arg residues in the sequence VRRFPWWWPFLRR make this a Trp-rich cationic peptide. The structure of tritrpticin bound to membrane-mimetic sodium dodecyl sulfate micelles has been determined using conventional two-dimensional NMR methods. It forms two adjacent turns around the two Pro residues, a distinct fold for peptide-membrane interaction. The first turn involves residues 4-7, followed immediately by a second well-defined 3(10)-helical turn involving residues 8-11. The hydrophobic residues are clustered together and are clearly separated from the basic Arg residues, resulting in an amphipathic structure. Favorable interactions between the unusual amphipathic fold and the micelle surface are probably key to determining the peptide structure. NMR studies of the peptide in the micelle in the presence of the spin-label 5-doxylstearic acid determined that tritrpticin lies near the surface of the micelle, where its many aromatic side chains appear to be equally partitioned into the hydrophilic-hydrophobic interface. Additional fluorescence studies confirmed that the tryptophan residues are inserted into the micelle and are partially protected from the effects of the soluble fluorescence quencher acrylamide.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable α-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable alpha-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Structure-function analysis of tritrpticin analogs: potential relationships between antimicrobial activities, model membrane interactions, and their micelle-bound NMR structures

Tritrpticin is a member of the cathelicidin family of antimicrobial peptides. Starting from its native sequence (VRRFPWWWPFLRR), eight synthetic peptide analogs were studied to investigate the roles of specific residues in its biological and structural properties. This included amidation of the C-terminus paired with substitutions of its cationic and Phe residues, as well as the Pro residues that are important for its two-turn micelle-bound structure. These analogs were determined to have a significant antimicrobial potency. In contrast, two other peptide analogs, those with the three Trp residues substituted with either Phe or Tyr residues are not highly membrane perturbing, as determined by leakage and flip-flop assays using fluorescence spectroscopy. Nevertheless the Phe analog has a high activity; this suggests an intracellular mechanism for antimicrobial activity that may be part of the overall mechanism of action of native tritrpticin as a complement to membrane perturbation. NMR experiments of these two Trp-substituted peptides showed the presence of multiple conformers. The structures of the six remaining Trp-containing analogs bound to dodecylphosphocholine micelles showed major, well-defined conformations. These peptides are membrane disruptive and show a wide range in hemolytic activity. Their micelle-bound structures either retain the typical turn-turn structure of native tritrpticin or have an extended alpha-helix. This work demonstrates that closely related antimicrobial peptides can often have remarkably altered properties with complex influences on their biological activities.     

Diversity of antimicrobial peptides and their mechanisms of action

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.     

Comparative In-Vitro Functional Analysis of Synthetic Defensins and Their Corresponding Peptide Variants Against HIV-1NL4.3, E. coli, S. aureus and P. aeruginosa

Defensins found in mammals belong to mainly two subfamilies α- and β-defensins. Mammalian defensins are small molecules (18–45 residues) that are cysteine, arginine rich compounds. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. To investigate the structure and activity relationship, amino acid substitutions that alter charge were introduced into synthetic defensin peptides by adding 2–2 Arg (RR) and Asp (DD) at both the terminal and tested their effects on HIV-1, E. coli, S. aureus, and P. aeruginosa. In the present study, we have chemically synthesized native defensin peptides and their variants with Arg (RR) and Asp (DD) amino acid residues at N- and C-termini. Later, we assayed their anti-HIV, anti-microbial activities, stability, cytotoxicity and hemolytic properties. We reported that anti-HIV and antimicrobial activities of native defensins is increased significantly by adding Arg (RR) residues at both the termini while the substitution of Arg (RR) with Asp (DD), eliminate anti-HIV and antimicrobial activity against all bacterial species tested. While other physical features i.e. stability, cell toxicity and hemolytic property were not affected by any of the changes in the sequence. The results suggest that the terminal residues in defensins are crucial functional elements that determine their microbicidal potency. The enhanced microbicidal activity observed for defensin peptides with Arg (RR) residues could be due to optimization of amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.     

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

The role of the abundant phenylalanines in the mode of action of the antimicrobial peptide clavanin

Clavanin A is a special antimicrobial peptide that acts at the level of the membrane via a pH-dependent mechanism. At neutral pH, clavanin disrupts biological and model membranes in a nonspecific manner, causing efflux of large molecules. At mildly acidic conditions, however, the peptide efficiently kills bacteria by permeabilizing their membrane most likely by interacting with proteins involved in proton translocation [Biochemistry 41 (2002) 7529]. Clavanin A is unusually rich in phenylalanines with 5 out of 23 residues, which suggests that these residues are functionally important. A set of mutants, in which all Phe residues are replaced by either Ile, Leu, Trp, or Tyr was used to investigate the role of these amino acids. The antimicrobial activities of the different peptides both at neutral and low pH show that the presence of phenylalanine is not essential nor optimal, as the Trp, Leu, and Ile mutant are equally or more active than the wild-type component. In general, at neutral pH, the biological activities correlate well with the peptides' ability to interact with membrane lipids. Correspondingly, the permeabilization efficiencies of biological and model membranes of the various derivatives were found to be closely related to their ability to adopt alpha-helical structures, and follows the order 5L>5W>5I>5Y>wild type. The results suggest an important role for the Phe residues, in providing the peptide in a balanced manner with sufficient hydrophobicity, and therewith membrane affinity, as well as conformational flexibility.     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.     

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.     

Partitioning of peptides and recombinant protein–peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structure

Genetic engineering has been used for fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in an aqueous two-phase system. The system was composed of dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer, EO30PO70. Peptides containing tryptophan, proline, arginine or aspartate residues were fused at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale extraction. The target protein and peptide tags were partitioned separately and then together in the fusion proteins in order to gain increased understanding of the influence of certain amino acid residues on the partitioning. The salt K₂SO₄ was used to reduce the charge dependent salt effects on partitioning and to evaluate the contribution to the partition coefficient from the hydrophobic–hydrophilic properties of the amino acid residues. The effect of Trp on peptide partitioning was independent of the difference in primary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only determined by the number of Trp. The effect of the charged residues, Arg and Asp, was dependent on the surrounding residues, i.e. if they were situated next to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fusion proteins. The effect of the salts sodium perchlorate and triethylammonium phosphate on the partitioning was also studied. The salt effects observed for the peptides were qualitatively similar to the effects observed for the fusion proteins.