Structure of amyloid beta fragments in aqueous environments

Conformational studies on amyloid beta peptide (Abeta) in aqueous solution are complicated by its tendency to aggregate. In this study, we determined the atomic-level structure of Abeta(28-42) in an aqueous environment. We fused fragments of Abeta, residues 10-24 (Abeta(10-24)) or 28-42 (Abeta(28-42)), to three positions in the C-terminal region of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). We then examined the structural properties in an aqueous environment. The host protein, Tk-RNase HII, is highly stable and the C-terminal region has relatively little interaction with other parts. CD spectroscopy and thermal denaturation experiments demonstrated that the guest amyloidogenic sequences did not affect the overall structure of the Tk-RNase HII. Crystal structure analysis of Tk-RNase HII(1-197)-Abeta(28-42) revealed that Abeta(28-42) forms a beta conformation, whereas the original structure in Tk-RNase HII(1-213) was alpha helix, suggesting beta-structure formation of Abeta(28-42) within full-length Abeta in aqueous solution. Abeta(28-42) enhanced aggregation of the host protein more strongly than Abeta(10-24). These results and other reports suggest that after proteolytic cleavage, the C-terminal region of Abeta adopts a beta conformation in an aqueous environment and induces aggregation, and that the central region of Abeta plays a critical role in fibril formation. This study also indicates that this fusion technique is useful for obtaining structural information with atomic resolution for amyloidogenic peptides in aqueous environments.     

Tilted properties of the 67-78 fragment of alpha-synuclein are responsible for membrane destabilization and neurotoxicity

Alpha-synuclein is a 140 residue protein associated with Parkinson's disease. Intraneural inclusions called Lewy bodies and Lewy neurites are mainly composed of alpha-synuclein aggregated into amyloid fibrils. Other amyloidogenic proteins, such as the beta amyloid peptide involved in Alzheimer's disease and the prion protein (PrP) associated with Creuztfeldt-Jakob's disease, are known to possess "tilted peptides". These peptides are short protein fragments that adopt an oblique orientation at a hydrophobic/hydrophilic interface, which enables destabilization of the membranes. In this paper, sequence analysis and molecular modelling predict that the 67-78 fragment of alpha-synuclein is a tilted peptide. Its destabilizing properties were tested experimentally. The alpha-synuclein 67-78 peptide is able to induce lipid mixing and leakage of unilamellar liposomes. The neuronal toxicity, studied using human neuroblastoma cells, demonstrated that the alpha-synuclein 67-78 peptide induces neurotoxicity. A mutant designed by molecular modelling to be amphipathic was shown to be significantly less fusogenic and toxic than the wild type. In conclusion, we have identified a tilted peptide in alpha-synuclein, which could be involved in the toxicity induced during amyloidogenesis of alpha-synuclein.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.     

Are the fusion processes involved in birth, life and death of the cell depending on tilted insertion of peptides into membranes?

Various peptide segments have been modeled as asymmetric amphipathic alpha-helices. Theoretical calculations have shown that they insert obliquely into model membranes. They have been named "tilted peptides". Molecular modeling results reported here also evidence the presence of tilted peptides in ADM-1 protein of Caenorhabditis elegans that may be involved in fusion events, in meltrin alpha, a protein implicated in myoblast fusion, in hemagglutinin of influenza virus, in the E2 glycoprotein of rubella virus, in the S protein of hepatitis B virus, in a subdomain of Ebola virus and in the malaria CS protein. Experimental results have indicated that tilted peptide fragments may be involved in cellular life events like sperm-egg fecondation, muscle development, protein translocation through signal sequences and cellular death caused by viral infection or parasite infestation. We speculate that membrane destabilization by these tilted peptides may be an important common step in life processes involving fusion phenomena.     

The crystal structure of a Dcp-containing peptide

We have investigated the conformational preferences of a newly synthesized C(alpha,alpha) symmetrically disubstituted glycine, namely alpha,alpha-dicyclopropylglycine (Dcp). We report here the crystal structure of a fully protected dipeptide containing Dcp, namely Z-Dcp(1)-Dcp(2)-OCH(3). Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an alpha-pleated sheet conformation, similar to that observed in linear peptides made up of alternating D- and L-residues and in Z-Aib-Aib-OCH(3) (Aib: alpha,alpha-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as molecular tool to stabilize folded conformations.     

The cisproline(i - 1)-aromatic(i) interaction: folding of the Ala-cisPro-Tyr peptide characterized by NMR and theoretical approaches

Cisproline(i - 1)-aromatic(i) interactions have been detected in several short peptides in aqueous solution by analysis of anomalous chemical shifts measured by 1H-NMR spectroscopy. This formation of local structure is of importance for protein folding and binding properties. To obtain an atomic-detail characterisation of the cisproline(i - 1)-aromatic(i) interaction in terms of structure, energetics and dynamics, we studied the minimal peptide unit, blocked Ala-cisPro-Tyr, using computational and experimental techniques. Structural database analyses and a systematic search revealed two groups of conformations displaying a cisproline(i - 1)-aromatic(i) interaction. These conformations were taken as seeds for molecular dynamics simulations in explicit solvent at 278 K. During a total of 33.6 ns of simulation, all the 'folded' conformations and some 'unfolded' states were sampled. 1H- and 13C-chemical shifts and 3J-coupling constants were measured for the Ala-Pro-Tyr peptide. Excellent agreement was found between all the measured and computed NMR properties, showing the good quality of the force field. We find that under the experimental and simulation conditions, the Ala-cisPro-Tyr peptide is folded 90% of the time and displays two types of folded conformation which we denote 'a' and 'b'. The type a conformations are twice as populated as the type b conformations. The former have the tyrosine ring interacting with the alanine alpha proton and are enthalpically stabilised. The latter have the aromatic ring interacting with the proline side chain and are entropically stabilised. The combined and complementary use of computational and experimental techniques permitted derivation of a detailed scenario of the 'folding' of this peptide.     

The N-terminal 12 residue long peptide of HIV gp41 is the minimal peptide sufficient to induce significant T-cell-like membrane destabilization in vitro

Here, we predicted the minimal N-terminal fragment of gp41 required to induce significant membrane destabilization using IMPALA. This algorithm is dedicated to predict peptide interaction with a membrane. We based our prediction of the minimal fusion peptide on the tilted peptide theory. This theory proposes that some protein fragments having a peculiar distribution of hydrophobicity adopt a tilted orientation at a hydrophobic/hydrophilic interface. As a result of this orientation, tilted peptides should disrupt the interface. We analysed in silico the membrane-interacting properties of gp41 N-terminal peptides of different length derived from the isolate BRU and from an alignment of 710 HIV strains available on the Los Alamos National Laboratory. Molecular modelling results indicated that the 12 residue long peptide should be the minimal fusion peptide. We then assayed lipid-mixing and leakage of T-cell-like liposomes with N-terminal peptides of different length as first challenge of our predictions. Experimental results confirmed that the 12 residue long peptide is necessary and sufficient to induce membrane destabilization to the same extent as the 23 residue long fusion peptide. In silico analysis of some fusion-incompetent mutants presented in the literature further revealed that they cannot insert into a modelled membrane correctly tilted. According to this work, the tilted peptide model appears to explain at least partly the membrane destabilization properties of HIV fusion peptide.     

Empty and peptide-loaded class II major histocompatibility complex proteins produced by expression in Escherichia coli and folding in vitro

The human class II major histocompatibility complex protein HLA-DR1 has been expressed in Escherichia coli as denatured alpha and beta subunits and folded in vitro to form the native structure. DR1 folding yields are 30-50% in the presence or absence of tight-binding antigenic peptides. The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight. It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociation and to proteolysis as does the native protein. The observed peptide specificity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein. We conclude that the recombinant DR1 has adopted the native fold. We have folded DR1 in the absence of peptide and isolated a soluble, peptide-free alphabeta-heterodimer. The empty DR1 can bind antigenic peptide but exhibits altered far UV-circular dichroism and thermal denaturation relative to the peptide-bound form.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

The construction of new proteins: V. A template-assembled synthetic protein (TASP) containing both a 4-helix bundle and beta-barrel-like structure

The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a beta-barrel as two folding "domains" is described. For the de novo design of proteins, amphiphilic helices (alpha) and beta-sheets (beta) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intramolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4 alpha)(4 beta). The design of this new macromolecule was assisted by computer modeling, which suggested a low-energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid-phase synthesis of the "two-domain" TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.     

Local and nonlocal environments around Cis peptides

Although the vast majority of peptide bonds in folded proteins are found in the trans conformation, a small percentage are found in the less energetically favorable cis conformation. Though the mechanism of cis peptide bond formation remains unknown, the role of local aromatics has been emphasized in the literature. This paper presents results from a comprehensive statistical analysis of both the local and nonlocal (i.e., tertiary) environment around cis peptides. In addition to an increased frequency of aromatic residues in the local environment around cis peptides, a number of nonlocal differences in protein secondary and tertiary structure between cis and trans peptides are found: (i) coil regions containing cis peptides are almost twice as long as those without cis peptides and include more Tyr and Pro residues; (ii) cis peptides occur with high frequencies in coil regions near large beta-structures; (iii) there is a nonlocal enrichment of Cys, His, Tyr, and Ser in the tertiary environment surrounding cis peptides when compared to trans peptides; and (iv) on average, cis peptides make fewer medium-range and more long-range contacts than trans peptides do. On the basis of these observations, it is concluded that nonlocal factors play a significant role in cis peptide formation, which has not been fully appreciated previously. An autocatalytic model for cis peptide formation is discussed as are consequences for protein folding.     

"De novo" design of peptides with specific lipid-binding properties

In this study, we describe an in silico method to design peptides that can be made of non-natural amino acids and elicit specific membrane-interacting properties. The originality of the method holds in the capacities developed to design peptides from any non-natural amino acids as easily as from natural ones, and to test the structure stability by an angular dynamics rather than the currently-used molecular dynamics. The goal of this study was to design a non-natural tilted peptide. Tilted peptides are short protein fragments able to destabilize lipid membranes and characterized by an asymmetric distribution of hydrophobic residues along their helix structure axis. The method is based on the random generation of peptides and their selection on three main criteria: mean hydrophobicity and the presence of at least one polar residue; tilted insertion at the level of the acyl chains of lipids of a membrane; and conformational stability in that hydrophobic phase. From 10,000,000 randomly-generated peptides, four met all the criteria. One was synthesized and tested for its lipid-destabilizing properties. Biophysical assays showed that the "de novo" peptide made of non-natural amino acids is helical either in solution or into lipids as tested by Fourier transform infrared spectroscopy and is able to induce liposome fusion. These results are in agreement with the calculations and validate the theoretical approach.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

Conformational effects of C(alpha,alpha)-dipropargylglycine as a constrained residue

A useful synthon to approach artificial phenylalanyl peptides in a [2 + 2 + 2] cycloaddition reaction, C(alpha,alpha)-dipropargylglycine (Dprg) is examined for its conformational preferences as a constrained residue. Crystal structure analysis and preliminary NMR results establish possible preference of the residue for folded (alpha) rather than extended (beta) region of the straight phi,psi conformational space. Boc-Dprg-L-Leu-OMe (1) displays two molecular conformations within the same crystallographic asymmetric unit, with Dprg in the alpha(R) or alpha(L) conformation, participating in a type I beta-turn or an alpha(L)-alpha(R)-type fold, in which Leu(2) assumes the alpha(R) conformation stereochemically favored for an L-chiral residue. Boc-Dprg-D-Val-L-Leu-OMe (2) displays a type I' beta-turn conformation in crystal, with both Dprg(1) and D-Val(2) assuming the alpha(L) conformation stereochemically favored for a D-chiral residue, with 4 --> 1 type hydrogen bond linking L-Leu(3) NH with Boc CO. NMR analysis using temperature variation, solvent titration, and a spin probe study suggests a fully solvent-exposed nature of Dprg NH, ruling out a fully extended C(5)-type conformation for this residue, and solvent sequestered nature of L-Leu(3) NH, suggesting possibility of a beta-turn due to Dprg assuming a folded conformation.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

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.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Studies of the erythrocyte spectrin tetramerization region

Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T. A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.     

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.     

Modeling the third loop of short-chain snake venom neurotoxins: roles of the short-range and long-range interactions

The influence of long-range interactions on local structures is an important issue in understanding protein folding process and protein structure stability. Using short-chain snake venom neurotoxin as a model system, we have studied the conformational properties of eight different loop III sequences either in the environment of one of the short-chain neurotoxin, erabutoxin b (PDB ID 1nxb), or in free state by Monte Carlo simulated annealing method. The surrounding protein structure was found to be crucial in stabilizing the loop conformation. Although all the eight peptides prefer type V beta turn in solution, three of them (KPGI, KPGV, KSGI) turn to type II beta turn and the other five (KKGI, KKGV, KNGI, KQGI, and KRGV) are confined to more rigid type V beta turn conformation in the protein structure. Using flexible tetra-glycine-peptide to screen the backbone conformational space in the protein environment also validates the results. This study shows that long-range interactions do contribute to the stability and the types of conformation for a surface loop in protein, while short-range interactions may only provide candidate conformations, which then have to be filtered by the long-range interactions further.