Antifreeze protein from shorthorn sculpin: identification of the ice-binding surface.

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.

Conformation and other biophysical properties of cyclic antimicrobial peptides in aqueous solutions.

As a step towards understanding the mechanism of the biological activity of cyclic antimicrobial peptides, the biophysical properties and conformations of four membrane-active cyclic peptide antibiotics, based on gramicidin S (GS), were examined in aqueous environments. These cyclic peptides, GS10 [cyclo(VKLdYP)2], GS12 [cyclo(VKLKdYPKVKLdYP)], GS14 [cyclo(VKLKVdYPLKVKLdYP)] and [d-Lys]4GS14 [cyclo(VKLdKVdYPLKVKLdYP)] (d-amino acid residues are denoted by d and are underlined) had different ring sizes of 10, 12 and 14 residues, were different in structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. GS10, GS12 and [d-Lys]4GS14 were shown to be monomeric in buffer systems with ionic strength biological environments. GS14 was also monomeric at low concentrations, but aggregated at concentrations > 50 microm. The affinity of peptides for self-assembly and interaction with hydrophobic surfaces was related to their free energy of intermolecular interaction. The effects of variations in salt and organic solvent (trifluoroethanol) concentration and temperature on peptide conformation were also examined. Similar to GS, GS10 proved to have a stable and rather rigid conformation in different environments and over a broad range of temperatures, whereas GS12, GS14 and [d-Lys]4GS14 had more flexible conformations. Despite its conformational similarity to GS10, GS14 had unique physicochemical properties due to its tendency to aggregate at relatively low concentrations. The biophysical data explain the direct relation between structure, amphipathicity and hydrophobicity of the cyclic peptides and their hemolytic activity. However, this relation with the antimicrobial activity of the peptides is of a more complex nature due to the diversity in membrane structures of microorganisms.

Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR.

The antimicrobial properties of the cyclic beta-sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state 19F-NMR was used to study a 19F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two 19F-labels was measured from their homonuclear dipolar coupling as 6 A. in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the 19F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the 19F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic beta-sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the 19F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the 19F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.

Diastereoisomeric analogues of gramicidin S: structure, biologicalactivity and interaction with lipid bilayers.

Analogues of a structurally equivalent version of theantimicrobial decameric cyclic peptide gramicidin S, GS10 [cyclo-(Val-Lys-Leu-d-Tyr-Pro)(2)], were designed to study theeffect of distortion in the beta-sheet/beta-turn structure of thecyclic peptide on its biological activity. In one approach, thehydrophobic nature of GS10 was conserved, and single amino acids in itsbackbone were replaced systematically with their correspondingenantiomers to give five diastereoisomeric analogues. In a relatedapproach, a more basic and hydrophilic analogue of GS10 [cyclo-(Lys-Val-Lys-d-Tyr-Pro(5)-Lys-Leu-Lys-d-Tyr-Pro(10))], together with two of itsmonosubstituted diastereoisomeric analogues (featuring d-Lys(1) or d-Val(2) respectively), weresynthesized. CD spectra were measured in a variety of environments,i.e. aqueous, aqueous trifluoroethanol and those containing SDSmicelles or phospholipid vesicles. In comparison with GS10 spectra, CDspectra of both groups of analogues in these environments exhibitedstructural distortion. Moreover, compared with GS10, antimicrobial andhaemolytic activities of the analogues were drastically decreased, implying the existence of a threshold minimum amphipathicity foreffective biological activity. However, in both groups of analogues,there was a correlation between amphipathicity and antimicrobial andhaemolytic activities. In the second group of analogues, bothelectrostatic and hydrophobic factors were related to theirantimicrobial and haemolytic activities. In order to gain an insightinto the nature of the biological activity of the two classes of cyclicpeptides, the relationship of their structure to interaction with lipidmembranes, and the implied mechanisms, were analysed in some detail inthe present study.

Development of the structural basis for antimicrobial and hemolytic activities of peptides based on gramicidin S and design of novel analogs using NMR spectroscopy.

The structures of 14-residue head-to-tail cyclic gramicidin S peptides have been investigated to develop the structural rationale for their antimicrobial and hemolytic profiles. The basis for these studies is GS14 (cyclo(VKLKVdYPLKVKLdYP)), designed as an extension of the naturally occurring antimicrobial peptide. The structure of GS14 has been determined using NMR methods and was found to exist in a highly amphipathic antiparallel beta-sheet conformation. Systematic enantiomeric substitutions within the framework of the GS14 peptide were found to decrease the amphipathicity of this molecule. These results indicated that there was a direct correlation between the high amphipathic character and potent hemolytic activity in the diastereomers, whereas an inverse correlation existed between amphipathicity and antimicrobial function. To define the structural consequences of changing the amphipathic nature of GS14 analogs to maximize antimicrobial activity and to minimize hemolysis, NMR structures were determined in water and the membrane-mimetic solvent trifluoroethanol. The structures show that these attributes are the result of induction of the beta-sheet character in a membrane environment and the positioning of charged side chains on the hydrophobic face of the cyclic framework, thus decreasing the amphipathicity and directed hydrophobicity of these molecules. Implications for the design of more effective antimicrobials are discussed.

New ice-binding face for type I antifreeze protein.

Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single alpha-helix that contains three 11 amino acid repeats (Thr-X(2)-Asx-X(7)), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully alpha-helical and remained as monomers in solution. Wild-type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild-type activity and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich surface and adjacent Thr.

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes.

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity.

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes.

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

Influence of preformed alpha-helix and alpha-helix induction on the activity of cationic antimicrobial peptides.

One prominent class of cationic antibacterial peptides comprises the alpha-helical class, which is unstructured in free solution but folds into an amphipathic alpha-helix upon insertion into the membranes of target cells. To investigate the importance of alpha-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form alpha-helices or to have preformed alpha-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i + 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized alpha-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of alpha-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase alpha-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r2 = 0.88) with a helican parameter which was calculated as the induction of alpha-helix in a membrane-mimicking environment divided by the alpha-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.

Unusual beta-sheet periodicity in small cyclic peptides.

Cyclic peptide homologs of gramicidin S containing 6, 8, 10, 12, 14 and 16 residues were synthesized and characterized using circular dichroism (CD) and 1H NMR spectroscopy. Based on the three-dimensional structures generated from these data we have found strong evidence of a periodic sequence-length dependence on beta-sheet content. In particular, peptides of length 6, 10 and 14 residues exhibit a high beta-sheet content, while peptides of 8, 12 and 16 residues appear to exist as random coils. This unusual beta-sheet periodicity may have important implications in our understanding of beta-sheet formation and in the design of constrained beta-sheet and beta-hairpin mimics.

Nonlamellar phases induced by the interaction of gramicidin S with lipid bilayers. A possible relationship to membrane-disrupting activity.

The interactions of the cyclic peptide gramicidin S (GS) with a variety of single-component lipid bilayers, and with membrane polar lipid extracts of Acholeplasma laidlawii B and Escherichia coli, were examined by differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. The DSC data indicate that the effects of GS on the thermotropic phase behavior of phosphatidylcholine and phosphatidylethanolamine dispersions are compatible with those expected of peptides interacting primarily with the polar headgroup and/or the polar/apolar interfaces of lipid bilayers. These DSC studies also suggest that GS exhibits stronger interactions with the more fluid bilayers. For mixtures of GS with lipids such as phosphatidylcholine, phosphatidylserine, cardiolipin, and sphingomyelin, axially symmetric 31P-NMR powder patterns are observed throughout the entire temperature range examined (0-90 degrees C), and there is little evidence for significant destabilization of the lipid bilayer with respect to nonlamellar phases. With mixtures of GS with either phosphatidylethanolamine, phosphatidylglycerol, or a nonlamellar phase-forming phosphatidylcholine, axially symmetric 31P-NMR powder patterns are also observed at low temperatures. However, at high temperatures, an isotropic component is observed in their 31P-NMR spectra, and the relative intensity of this component increases significantly with temperature and with GS concentration. Once formed at high temperatures, this isotropic component exhibits a marked cooling hysteresis and in most cases disappears only when the sample is recooled to temperatures well below the lipid hydrocarbon chain-melting phase transition temperature. We also show that GS induces the formation of isotropic components in the 31P-NMR spectra of heterogeneous lipid mixtures such as occur in A. laidlawii B and E. coli membranes. These observations suggest that GS induces the formation of cubic or other three dimensionally ordered inverted nonlamellar phases when it interacts with some types of lipid bilayers, a suggestion strongly supported by our X-ray diffraction studies. Our results also suggest that the capacity of GS to induce the formation of such phases increases with the intrinsic nonlamellar phase-preferring tendencies of the lipids with which it interacts probably by producing localized increases in membrane monolayer curvature stress. The latter effect could be part of the mechanism through which this peptide exhibits its antimicrobial and hemolytic activities.

Modulation of structure and antibacterial and hemolytic activity by ring size in cyclic gramicidin S analogs.

We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in beta-sheet content, with rings containing 6, 10, and 14 residues exhibiting beta-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Gram-positive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gram-negative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.

Gramicidin S is active against both gram-positive and gram-negative bacteria.

Four linear and four cyclic analogs of gramicidin S (GS) in which D-Phe was replaced with either D-His, D-Ser, D-Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 micrograms/mL for gram-negative bacteria, compared to MICs of 3 micrograms/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the D-Phe to D-Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes.

Preferential heterodimeric parallel coiled-coil formation by synthetic Max and c-Myc leucine zippers: a description of putative electrostatic interactions responsible for the specificity of heterodimerization.

The oncoprotein c-Myc must heterodimerize with Max to bind DNA and perform its oncogenic activity. The c-Myc-Max heterodimer binds DNA through a basic helix-loop-helix leucine zipper (b-HLH-zip) motif and it is proposed that leucine zipper domains could, in concert with the HLH regions, provide the specificity and stability of the b-HLH-zip motif. In this context, we have synthesized the peptides corresponding to the leucine zipper domains of Max and c-Myc with a N-terminal Cys-Gly-Gly linker and studied their dimerization behavior using reversed-phase HPLC and CD spectroscopy. The preferential formation of a fully helical parallel c-Myc-Max heterodimeric coiled-coil was observed under air-oxidation and redox conditions at neutral pH. We show that the stability and the helicity of the disulfide-linked c-Myc-Max heterostranded coiled-coil is modulated by pH, with a maximum around pH 4.5, supporting the existence of stabilizing and specific interhelical electrostatic interactions. We present a molecular model of the c-Myc-Max heterostranded coiled-coil describing potential electrostatic interactions responsible for the specificity of the interaction, the main feature being putative buried electrostatic interactions between a histidine side-chain (in the Max leucine zipper) and two glutamic acid side-chains (in the c-Myc leucine zipper) at the heterodimer interface. This model is supported by the fact that the apparent pKa (as determined by [1H]-NMR spectroscopy) of this histidine side-chain at 25 degrees C is 0.42 (+/- 0.05) pKa units higher in the folded form than in the unfolded form. This indicates that the charged histidine side-chain contributes approximately 0.57 (+/- 0.07) kcal/mol (2.38 (+/- 0.30) kJ/mol) of stabilization free energy to the c-Myc-Max heterostranded coiled-coil through favorable electrostatic interaction.

Synthesis and characterization of carbohydrate-linked murine monoclonal antibody K20-human serum albumin conjugates.

Efficacy of antibody mediated targeting depends on retention of immunoreactivity in conjugates. Retention can be improved by site-specific linkage of drugs or drug-loaded carriers to residues that are located well away from the antigen-binding sites. In this study we describe the site-specific linkage of a potential drug carrier, human serum albumin (HSA), to the carbohydrate residues in Dal K20, a murine IgG1 monoclonal antibody (mAb) against human renal cell carcinoma, using disulfide exchange between 3-(2-pyridyldithio)propionic acid succinimidyl ester (SPDP)-derivatized HSA and 11-[[3-(2-pyridyldithio)propionyl]amino]undecanoic acid hydrazide (AUPDP)-derivatized mAb Dal K20. AUPDP gave a higher yield of the conjugate than a functionally analogous 3-(2-pyridyldithio)propionic acid hydrazide (HPDP), suggesting that the extra length of the former facilitated the linkage. The conjugates were found to be unstable without reduction of the hydrazone linkage using sodium cyanoborohydride. Stabilized 1:1 HSA:K20 carbohydrate-linked conjugates were isolated and compared with non-site-specific 1:1 conjugates in which HSA was conjugated to amino groups in mAb Dal K20. The yield and stability of the two conjugates were comparable, but the site-specific conjugate was found to retain three times more antibody activity than the non-site-specific conjugate.