Characterization of the 4D5Flu single-chain antibody with a stimulus-responsive elastin-like peptide linker: a potential reporter of peptide linker conformation

Single-chain antibodies (scFvs) are comprised of IgG variable light and variable heavy domains tethered together by a peptide linker whose length and sequence can affect antigen binding properties. The ability to modulate antigen binding affinity through the use of environmental triggers would be of great interest for many biotechnological applications. We have characterized the antigen binding properties of an anti-fluorescein scFv, 4D5Flu, containing stimulus-responsive short elastin-like peptide linkers and nonresponsive flexible linkers. Comparison of length-matched flexible and short elastin-like peptide linkers indicates that a stimulus-responsive linker can confer stimulus-responsive control of fluorescein binding. A linker length of either six or 10 amino acids proved to have the largest thermally induced response. Similar differences in binding free energy changes indicate a common underlying mechanism of thermal responsiveness. Contrary to the thermal behavior, the effect of salt, another elastin beta-turn-inducing stimulus, stabilized antigen binding in the six- and 10-amino-acid linkers such that elastin-like linkers became less stimulus-responsive as compared with flexible linkers. Again, the thermodynamic analysis indicates a common mechanism of salt responsiveness. Characterization of the room-temperature binding affinities and evidence indicating a dimeric state of the scFvs concomitantly suggest the major contribution to the stimulus-responsive behavior derives from the perturbation of interdomain associations, rather than the linker-constrained disruption of the intramolecular association. The ability to use stimulus-responsive peptide modules to exert a novel control over protein function will likely find application in the creation of allosteric antibodies and scFv-based biosensors, and as a platform to enable the evolution of new stimulus-responsive peptides.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.     

Conformational properties of a peptide model for unfolded alpha-helices

Models of protein folding often hypothesize that the first step is local secondary structure formation. The assumption is that unfolded polypeptide chains possess an intrinsic propensity to form these local secondary structures. On the basis of this idea, it is tempting to model the local conformational properties of unfolded proteins using well-established residue secondary structure propensities, in particular, alpha-helix forming propensities. We have used spectroscopic methods to investigate the conformational behavior of a host-guest series of peptides designed to model unfolded alpha-helices. A suitable peptide model for unfolded alpha-helices was determined from studies of the length dependence of the conformational properties of alanine-based peptides. The chosen host peptide possessed a small, detectable, alpha-helix content. Substituting various representative guest residues into the central position of the host peptide at times changed the conformational behavior dramatically, and often in ways that could not be predicted from known alpha-helix forming propensities. The data presented can be used to rationalize some of these propensities. However, it is clear that secondary structure propensities cannot be used to predict the local conformational properties of unfolded proteins.     

Synthesis and conformation of a polyoxyethylene-bound undecapeptide of the alamethicin helix and (2-methylalanyl-L-alanine)1–7

The stepwise synthesis and conformational studies of the N-terminal helical partial sequence of the membrane-modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N-t-Boc-L -Pro-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH and N-Ac-Aib-L -Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α-helix formation of the N-protected peptide polymers beginning with the nonapeptide and of the N-protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature- and solvent-dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random-coil conformation. For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib-L -Ala)_n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right-handed α-helix with increasing peptide length. This change is observed for the N-protected peptides at a chain length of 8 residues and for the N-protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene-bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t-Boc-(Aib-L -Ala)_n-OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.     

Conformational properties of gastrin fragments of increasing chain length

The conformational properties of a series of biologically active gastrin peptides of increasing chain length have been investigated in TEE solution by spectroscopic techniques. It was found that elongation of the glutamic acid sequence from 1 to 5 residues at the N-terminal portion of the molecules causes a cooperative change of the conformation of the peptide backbone. The environment of the biologically important C-terminal sequence-Trp-Nle-Asp-Phe-NH₂ monitored by the near-uv chiroptical propertical properties is alos affected by chain elongation. However, the change of the structure of the C-terminal portion does not parallel the conformational change of the peptide backbone. In fact, the final folded structure at the C-terminus is almost reached in the fragment with a sequence of 4 glutamic acid residues, while an additiona, relevent conformational change of the backbne is observed on further elongation of the chain to minigastrin and little gastrin. The ability of the fragments to fold into an ordered conformation on chain elongation parallels the increase of biolocical potency tested in vivo, reported in the literature, and suggests a correlation between these two facts. Ionization of the carboxyl side chains is without effect on the structure of the fragments with 2, 3, and 4 glutamic acid residues, while an effect is observed in minigastrin and little gastrin. From analysis of the CD properties and from their dependence upon side-chain ionization a structural model is proposed for the hormones minigastrin and little gastrin. This tentative model includes a β-bend located in the sequence Ala-Tyr-Gly-Trp- and a short helical section at the N-terminal portion of the hormones.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylcholine bilayers

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of an alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and odd-chain members of the homologous series of n-saturated diacylphosphatidylcholines. An analogue of L24, in which the lysine residues were all replaced by 2,3-diaminopropionic acid, and another, in which a leucine residue at each end of the polyLeu sequence was replaced by a tryptophan, were also studied. At low peptide concentrations, the DSC thermograms exhibited by these lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the transition temperature and cooperativity of this component, and its fractional contribution to the total enthalpy change, decrease with an increase in peptide concentration, more or less independently of phospholipid acyl chain length. The other component is very broad and predominates at high peptide concentrations. These two components have been assigned to the chain-melting phase transitions of populations of peptide-poor and peptide-enriched lipid domains, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that these peptides reduce somewhat but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the broad phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length. Our FTIR spectroscopic data indicate that these peptides form a very stable alpha-helix under all of our experimental conditions but that small distortions of their alpha-helical conformation are induced in response to mismatch between peptide hydrophobic length and gel-state bilayer hydrophobic thickness. We also present evidence that these distortions are localized to the N- and C-terminal regions of these peptides. Interestingly, replacing the terminal Lys residues of L24 by 2,3-diaminopropionic acid residues actually attenuates the hydrophobic mismatch effects of the peptide on the thermotropic phase behavior of the host PC bilayer, in contrast to the predictions of the snorkel hypothesis. We rationalize this attenuated hydrophobic mismatch effect by postulating that the 2,3-diaminopropionic acid residues are too short to engage in significant electrostatic and hydrogen-bonding interactions with the polar headgroups of the host phospholipid bilayer, even in the absence of any hydrophobic mismatch between incorporated peptide and the bilayer. Similarly, the reduced hydrophobic mismatch effect also observed when the two terminal Leu residues of L24 are replaced by Trp residues is rationalized by considering the lower energetic cost of exposing the Trp as opposed to the Leu residues to the aqueous phase in thin PC bilayers and the higher cost of inserting the Trp as opposed to the Leu residues into the hydrophobic cores of thick PC bilayers.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Lipid-induced conformational changes in glucagon,secretin, and vasoactive intestinal peptide

The CD of glucagon, secretin, and vasoactive intestinal peptide has been studied as a function of temperature in water and in aqueous solutions of dodecyl sulfate, phosphatidyl glycerol, and L -α-phosphatidic acid (dipalmitoyl). The anionic detergent and lipids induce helix formation in all three peptides, with the amount of induced helical content increasing in the order glucagon < secretin < vasoactive intestinal peptide. These observations are subject to quantitative rationalization using a matrix formulation for the configuration partition function. In this formulation the major conformational consequences of the interaction with anionic lipids or detergents is an increase in the probability for helix formation by arginyl, histidyl, and lysyl residues. The region in which helix formation is maximal is found to be at amino acid residues 13–20 in all three peptides. Other studies have implicated this portion of the polypeptide chain in receptor binding. Thus, the helical segment induced by interaction with anionic lipids may play an important physiological role.     

Elastin-like Peptide amphiphiles form nanofibers with tunable length

Peptide amphiphiles (PAs) self-assemble nanostructures with potential applications in drug delivery and tissue engineering. Some PAs share environmentally responsive behavior with their peptide components. Here we report a new type of PAs biologically inspired from human tropoelastin. Above a lower critical solution temperature (LCST), elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition. Similar to other PAs, elastin-like PAs (ELPAs) assemble micelles with fiber-like nanostructures. Similar to ELPs, ELPAs have inverse phase transition behavior. Here we demonstrate control over the ELPAs fiber length and cellular uptake. In addition, we observed that both peptide assembly and nanofiber phase separation are accompanied by a distinctive secondary structure attributed primarily to a type-1 β turn. We also demonstrate increased solubility of hydrophobic paclitaxel (PAX) in the presence of ELPAs. Due to their biodegradability, biocompatibility, and environmental responsiveness, elastin-inspired biopolymers are an emerging platform for drug and cell delivery; furthermore, the discovery of ELPAs may provide a new and useful approach to engineer these materials into stimuli-responsive gels and drug carriers.     

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.     

Temperature-induced conformational transition of a model elastin-like peptide GVG(VPGVG)(3) in water

The conformation of a single elastin-like peptide GVG(VPGVG)3 in liquid water is studied by computer simulations in the temperature interval between 280 and 440 K. Two main conformational states of the peptide can be distinguished: a rigid conformational state, dominating at low temperatures, and a flexible conformational state, dominating at high temperatures. A temperature-induced transition between these states occurs at about 310 K, rather close to a transition temperature seen in experiments. This transition is accompanied by the thermal breaking of the hydrogen-bonded spanning network of the hydration water via a percolation transition upon heating. This finding indicates that the H-bond clustering structure of the hydration water plays an important role in the conformational stability of biomolecules. A second important observation is the Gaussian distribution of the end-to-end distance in the high-temperature state, which supports the idea of a rubber-like elasticity of the studied elastin-like peptide. Finally our results challenge the idea of the folding of elastin-like peptides upon heating.     

Three dimensional structure of the transmembrane region of the proto-oncogenic and oncogenic forms of the neu protein.

The neu proto-oncogene may be converted into a dominantly transforming oncogene by a single point mutation. Substitution of a valine residue at position 664 in the transmembrane region with glutamic acid activates the tyrosine kinase of the molecule and is associated with increased receptor dimerization. Previously we have proposed a model in which the glutamic acid side chain stabilizes receptor dimerization by hydrogen bonding. Other models have been proposed in which the mutation leads to a conformational change in the transmembrane region mimicking that assumed to occur following binding of a natural ligand. Synthetic peptides representing part of the transmembrane region were prepared. Some residues were replaced with serine in order to improve peptide solubility to allow purification and analysis. Both the peptides containing valine and glutamic acid dissolved in water and in an artificial lipid monolayer. The structures of the peptides were determined by NMR spectroscopy to be alpha-helical. No significant difference in conformation was observed between the two peptides. This result does not support the model proposing a conformational change. The receptor structures determined experimentally do allow alternative models involving receptor transmembrane region packing.     

Nucleophile specificity of subtilisin in an organic solvent with low water content: investigation via acyl transfer reactions

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, p(app), determined from the HPLC analysis of the reactions. The order of preference for the P'(1) position was estimated to be: Gly > hydrophilic, positively charged > hydrophobic, aromatic > negatively charged > Leu > Pro side chain. For the P'(2) position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of p(app) for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P'(3) position. The dipeptide with Pro in P'(1) did not react at all, but a tripeptide having Pro in P'(3) was a very good nucleophile. The negatively charged amino acid residues in the P'(1) position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P'(2) and P'(3) are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of alpha-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed. (c) 1996 John Wiley & Sons, Inc.     

Exploratory synthesis of peptide-alpha-thioester segments spanning the polypeptide sequence of the delta-opioid receptor, a G protein-coupled receptor

We have decided to use the delta-opioid receptor (372 residues) as a model system to develop methods for the total chemical synthesis of G protein-coupled receptors. The most important feature of this receptor from a chemical synthesis perspective is the wealth of cysteines spread throughout its sequence, which are required for native chemical ligation. A total of 13 cysteines are located in the the delta-opioid receptor polypetide chain in both loop and putative transmembrane (TM) regions. We envisioned a synthesis of the polypeptide that would make use of peptide-alpha-thioesters ranging from 37 to 63 residues in length. Here, we report data from an exploratory synthesis of such a set of peptide-alpha-thioesters. For all seven peptides, the crude material approximately 30 residues into the synthesis was sufficiently homogeneous to make isolation and purification straightforward. Extension of the peptides to between 40 and 50 residues in length generally produced a significant decrease in the quality of the crude products, although in most cases, we judged that high purity peptides could probably be isolated. By 60 residues, however, the crude peptide product mixtures are probably too heterogeneous to purify to homogeneity by reversed-phase HPLC. In general, delta-opioid receptor peptides with a single predicted TM domain were sufficiently soluble to handle postcleavage and to analyze by reversed-phase HPLC, whereas 1.5 TM domains rendered the peptides too hydrophobic to handle or analyze by standard protocols. Given the challenges of chain assembly, handling, and purification of these peptides, a synthetic strategy that uses approximately 12 or 13 shorter peptide segments of 20-40 residues each is probably a more feasible approach.     

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.     

Novel peptide-shelled dendrimer with dramatically changeable thermo-responsive character

The preparation of a novel peptide/dendrimer hybrid is reported in which an elastin-like oligopeptide is successfully assembled onto a poly(amidoamine) dendrimer surface (G4-ELP), and its unique thermo-responsive behavior is discussed. As a result, the G4-ELP is found to exhibit LCST behavior in the pH range 3-10 including physiological temperature range under neutral-pH conditions. Moreover, cooperative interplay between the folding state of the ELP shell and the ionization state of the dendrimer core enables the G4-ELP to control its LCST widely by pH variation. This achievement provides a new insight for the design of dual-responsive materials with a potential in biological applications.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.     

13C CPMAS NMR studies of the elastin-like polypeptide (LGGVG)n

The elucidation of structure-function relationships in insoluble elastin is often approached using elastin-like polypeptides. In this manner, the characterization of the different regions in this extensive biopolymer may be facilitated in a "piece-wise" manner. Our solid-state NMR experiments indicate that (LGGVG)n has structural similarities to elastin and some elastin peptides, providing support for the utility of the mimetic peptides. Furthermore, previous NMR and CD studies indicated that the structure of the elastin-like polypeptide (LGGVG)n in solution is best described as a "conformational ensemble" with a mixture of type I and II beta-turns, in addition to unfolded regions. Our data indicate that the peptide does not adopt a single conformation in the solid state, lending further support to models for elastin that involve significant conformational heterogeneity.     

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.