Dissection of a beta-barrel motif leads to a functional dimer: the case of the intestinal fatty acid binding protein.
ABSTRACT: A lingering issue in the area of protein engineering is the optimal design of beta motifs. In this regard, the framework provided by intestinal fatty acid binding protein (IFABP) was successfully chosen to explore the consequences on structure and function of the redesign of natural motifs. A truncated form of IFABP (Delta 98 Delta) served to illustrate the nonintuitive notion that the integrity of the beta-barrel can indeed be compromised with no effect on the ability to attain a native-like fold. This is most likely the outcome of the key role played by the preservation of essential core residues. In the search for the minimal structural determinants of this fold, Delta 98 Delta offered room for further intervention. A dissection of this protein leads to a new abridged variant, Delta 78 Delta, containing 60% of the amino acids of IFABP. Spectroscopic analyses indicate that Delta 78 Delta retains substantial beta-sheet content and preserves tertiary interactions, displaying cooperative unfolding and binding activity. Most strikingly, this construct adopts a remarkably stable dimeric structure in solution. This phenomenon takes advantage of the inherent structural plasticity of this motif, likely profitting from edge-to-edge interactions between beta-sheets, whereas avoiding the most commonly occurring outcome represented by aggregation.
Project description:The design of beta-barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on beta-barrel architecture and stability could be learned. In this context, Delta98Delta (encompassing residues 29-126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the beta-barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Delta98Delta adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the beta-barrel can indeed be compromised with no consequence on the ability to attain a native-like and functional fold.
Project description:A clear understanding of the structural foundations underlying protein aggregation is an elusive goal of central biomedical importance. A step toward this aim is exemplified by the ?-barrel motif represented by the intestinal fatty acid binding protein (IFABP) and two abridged all-? sheet forms (?98? and ?78?). At odds with the established notion that a perturbation of the native fold should necessarily favor a buildup of intermediate forms with an enhanced tendency to aggregate, the intrinsic stability (?G°H2O) of these proteins does not bear a straightforward correlation with their trifluoroethanol (TFE)-induced aggregation propensity. In view of this fact, we found it more insightful to delve into the connection between structure and stability under sub-aggregating conditions (10% TFE). In the absence of the co-solvent, the abridged variants display a common native-like region decorated with a disordered C-terminal stretch. Upon TFE addition, an increase in secondary structure content is observed, assimilating them to the parent protein. In this sense, TFE perturbs a common native like region while exerting a global compaction effect. Importantly, in all cases, fatty acid binding function is preserved. Interestingly, energetic as well as structural diversity in aqueous solution evolves into a common conformational ensemble more akin in stability. These facts reconcile apparent paradoxical findings related to stability and rates of aggregation. This scenario likely mimics the accrual of aggregation-prone species in the population, an early critical event for the development of fibrillation.
Project description:?98? is a functional all-? sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of ?-barrel proteins. Albeit displaying increased conformational plasticity, ?98? exhibits a nativelike ?-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and ?98? regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.
Project description:Intestinal fatty acid binding protein (IFABP) interacts with biological membranes and delivers fatty acid (FA) into them via a collisional mechanism. However, the membrane-bound structure of the protein and the pathway of FA transfer are not precisely known. We used molecular dynamics (MD) simulations with an implicit membrane model to determine the optimal orientation of apo- and holo-IFABP (bound with palmitate) on an anionic membrane. In this orientation, the helical portal region, delimited by the alphaII helix and the betaC-betaD and betaE-betaF turns, is oriented toward the membrane whereas the putative beta-strand portal, delimited by the betaB-betaC, betaF-betaG, betaH-betaI turns and the N terminus, is exposed to solvent. Starting from the MD structure of holo-IFABP in the optimal orientation relative to the membrane, we examined the release of palmitate via both pathways. Although the domains can widen enough to allow the passage of palmitate, fatty acid release through the helical portal region incurs smaller conformational changes and a lower energetic cost.
Project description:This study aimed to examine the source-level cortical brain networks of post-traumatic stress disorder (PTSD) based on the graph theory using electroencephalography (EEG). Sixty-six cortical source signals were estimated from 78 PTSD and 58 healthy controls (HCs) of resting-state EEG. Four global indices (strength, clustering coefficient (CC), path length (PL) and efficiency) and one nodal index (CC) were evaluated in six frequency bands (delta, theta, alpha, low beta, high beta and gamma). PTSD showed decreased global strength, CC and efficiency, in delta, theta, and low beta band and enhanced PL in theta and low beta band. In low beta band, the strength and CC correlated positively with the anxiety scores, while PL had a negative correlation. In addition, nodal CCs were reduced in PTSD in delta, theta and low beta band. Nodal CCs of theta band correlated negatively with rumination and re-experience symptom scores; while, nodal CCs in low beta band correlated positively with anxiety and pain severity. Inefficiently altered and symptom-dependent changes in cortical networks were seen in PTSD. Our source-level cortical network indices might be promising biomarkers for evaluating PTSD.
Project description:The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (</=2 mM) concentrations. Changes to the residues in the Hao-containing strands of 1c and 1g improve folding and show that folding of the structures can be enhanced without altering the sequence of the pentapeptide strand. Well-folded cyclic modular beta-sheets 1a, 1b, and 1f each have a phenylalanine directly across from Hao, suggesting that cyclic modular beta-sheets containing aromatic residues across from Hao are better folded.
Project description:This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.
Project description:Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.
Project description:A consensus classification and nomenclature are defined for RNA backbone structure using all of the backbone torsion angles. By a consensus of several independent analysis methods, 46 discrete conformers are identified as suitably clustered in a quality-filtered, multidimensional dihedral angle distribution. Most of these conformers represent identifiable features or roles within RNA structures. The conformers are given two-character names that reflect the seven-angle delta epsilon zeta alpha beta gamma delta combinations empirically found favorable for the sugar-to-sugar "suite" unit within which the angle correlations are strongest (e.g., 1a for A-form, 5z for the start of S-motifs). Since the half-nucleotides are specified by a number for delta epsilon zeta and a lowercase letter for alpha beta gamma delta, this modular system can also be parsed to describe traditional nucleotide units (e.g., a1) or the dinucleotides (e.g., a1a1) that are especially useful at the level of crystallographic map fitting. This nomenclature can also be written as a string with two-character suite names between the uppercase letters of the base sequence (N1aG1gN1aR1aA1cN1a for a GNRA tetraloop), facilitating bioinformatic comparisons. Cluster means, standard deviations, coordinates, and examples are made available, as well as the Suitename software that assigns suite conformer names and conformer match quality (suiteness) from atomic coordinates. The RNA Ontology Consortium will combine this new backbone system with others that define base pairs, base-stacking, and hydrogen-bond relationships to provide a full description of RNA structural motifs.
Project description:This paper describes the X-ray crystallographic structure of a designed cyclic beta-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics beta-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (1a) contains molecular template and turn units that induce beta-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two "Hao" amino acids that help template the beta-sheet structure and the two delta-linked ornithine turn units that link the Hao-containing template to the heptapeptide beta-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a beta-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide beta-strand. The delta-linked ornithines adopt a conformation that resembles a hydrogen-bonded beta-turn, in which the ornithine takes the place of the i+1 and i+2 residues. The dimers formed by macrocyclic beta-sheet 1a resemble the dimers of many proteins, such as defensin HNP-3, the lambda-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of 1a self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic beta-sheet peptides that were observed in solution.