Folding & design最新文献

Background: Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated.

Results: The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1–155 and 156–416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60–85%) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012 ± 0.0035 and 0.007 ± 0.0020 s⁻¹). A prior renaturation of the C-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step.

Conclusions:In addition to the previously observed slow refolding step (0.012 s⁻¹) within the C-domain, the occurrence of another slow step (0.007 s⁻¹), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.

Background: In vitro selection has been shown previously to be a powerful method for isolating nucleic acids with specific ligand-binding functions (‘aptamers’). Given this capacity, we have sought to isolate RNA motifs that can confer fluorescent labeling to tagged RNA transcripts, potentially allowing in vivo detection and in vitro spectroscopic analysis of RNAs.

Results: Two aptamers that recognize the fluorophore sulforhodamine B were isolated by the in vitro selection process. An unusually large motif of approximately 60 nucleotides is responsible for binding in one RNA (SRB-2). This motif consists of a three-way helical junction with two large, highly conserved unpaired regions. Phosphorothioate mapping with an iodoacetamide-tagged form of the ligand shows that these two regions make close contacts with the fluorophore, suggesting that the two loops combine to form separate halves of a binding pocket. The aptamer binds the fluorophore with high affinity, recognizing both the planar aromatic ring system and a negatively charged sulfonate, a rare example of anion recognition by RNA. An aptamer (FB-1) that specifically binds fluorescein has also been isolated by mutagenesis of a sulforhodamine aptamer followed by re-selection. In a simple in vitro test, SRB-2 and FB-1 have been shown to discriminate between sulforhodamine and fluorescein, specifically localizing each fluorophore to beads tagged with the corresponding aptamer.

Conclusions:In addition to serving as a model system for understanding the basis of RNA folding and function, these experiments demonstrate potential applications for the aptamers in transcript double labeling or fluorescence resonance energy transfer studies.

Background: Glycine is an intrinsically destabilizing residue in β sheets. In natural proteins, however, this destabilization can be ‘rescued’ by specific cross-strand pairing with aromatic residues. Here, we present an experimental study of this effect.

Results: Protein variants containing glycine and aromatic residues positioned across β strands in both antiparallel and parallel orientations were studied. The pairing of glycine and phenylalanine across antiparallel strands resulted in a synergistic increase in protein stability. Dramatic differences in stability were observed for the parallel β-sheet mutants, which were dependent upon the type of site occupied by glycine as well as the type of aromatic residue with which it was cross-strand paired.

Conclusions:Experimental results from a series of mutants suggest a thermodynamic benefit for glycine–aromatic pairing across antiparallel β strands, consistent with the prevalence of such pairs in natural proteins. We also demonstrate the specificity of glycine–aromatic interactions across parallel β strands, which defines strand register.

Systematic studies of kinetics using minimal protein models reveal multiple folding nuclei for sequences that reach the native state in a single step. The diversity of the folding nuclei depends on sequence and topology.

Background: Atomic level rotamer libraries for sidechains in proteins have been proposed by several groups. Conformations of side groups in coarse-grained models, on the other hand, have not yet been analyzed, although low resolution approaches are the only efficient way to explore global structural features.

Results: A residue-specific backbone-dependent library for sidechain isomers, compatible with a coarse-grained model, is proposed. The isomeric states are utilized in packing sidechains of known backbone structures. Sidechain positions are predicted with a root-mean-square deviation (rmsd) of 2.40 å with respect to crystal structure for 50 test proteins. The rmsd for core residues is 1.60 å and decreases to 1.35 å when conformational correlations and directional effects in inter-residue couplings are considered.

Conclusions:An automated method for assigning sidechain positions in coarse-grained model proteins is proposed and made available on the internet; the method accounts satisfactorily for sidechain packing, particularly in the core.

Background: The most conspicuous feature of a right-handed α helix is the presence of hydrogen bonds between the backbone carbonyl oxygen and NH groups along the chain. A simple off-lattice model that includes hydrogen bond interactions using virtual atoms is used to examine the stability, cooperativity and kinetics of the helix–coil transition.

Results: We have studied the thermodynamics (using multiple histogram method) and kinetics (by Brownian dynamics simulations) of 16-mer minimal off-lattice models of four-turn α-helix sequences. The carbonyl and NH groups are represented as virtual moieties located between two α-carbon atoms along the polypeptide chain. The characteristics of the native conformations of the model helices, such as the helical pitch and angular correlations, coincide with those found in real proteins. The transition from coil to helix is quite broad, which is typical of these finite-sized systems. The cooperativity, as measured by a dimensionless parameter, Ω _c, that takes into account the width and the slope of the transition curves, is enhanced when hydrogen bonds are taken into account. The value of Ω _c for our model is consistent with that inferred from experiment for an alanine-based helix-forming peptide. The folding time τ_F ranges from 6 to 1000 ns in the temperature range 0.7–1.9 T_F, where T_F is the helix–coil transition temperature. These values are in excellent agreement with the results from recent fast folding experiments. The temperature dependence of τ_F exhibits a nearly Arrhenius behavior. Thermally induced unfolding occurs on a time scale that is less than 40–170 ps depending on the final temperature. Our calculations also predict that, although τ_F can be altered by changes in the sequence, the dynamic range over which such changes take place is not as large as that predicted for β-turn formation.

Conclusions:Hydrogen bonds not only affect the stability of α-helix formation but also have profound influence on the kinetics. The excellent agreement between our calculations and experiments suggests that these models can be used to investigate the effects of sequence, temperature and viscosity on the helix–coil transition.

Background: Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures.

Results: The structural conservation and variation of the active sites of the α/β hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the α/β hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the α/β hydrolase family.

Conclusions:The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.