Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway

Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.     

Computational diagnosis of protein conformational diseases: short molecular dynamics simulations reveal a fast unfolding of r-LDL mutants that cause familial hypercholesterolemia

The molecular basis of conformational diseases frequently resides in mutant proteins constituting a subset of the vast mutational space. While the subtleties of protein structure point to molecular dynamics (MD) techniques as promising tools for an efficient exploration of such a space, the average size of proteins and the time scale of unfolding events make this goal difficult with present computational capabilities. We show here, nevertheless, that an efficient approach is already feasible for modular proteins. Familial hypercholesterolemia (FH) is a conformational disease linked to mutations in the gene encoding the low density lipoprotein receptor. A high percentage of these mutations has been found in the seven small modular binding repeats of the receptor. Taking advantage of its small size, we have performed an in depth MD study of the fifth binding repeat. Fast unfolding dynamics have been observed in the absence of a structural bound calcium ion, which agrees with its reported essential role in the stability of the module. In addition, several mutations detected in FH patients have been analyzed, starting from the native conformation. Our results indicate that in contrast with the wild type protein and an innocuous control mutant, disease-related mutants experience, in short simulation times (2-8 ns), gross departures from the native state that lead to unfolded conformations and, in some cases, to binding site desorganization deriving in calcium release. Computational diagnosis of mutations leading to conformational diseases seems thus feasible, at least for small or modular pathogenic proteins.     

Maltose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway

The hyperthermophilic anaerobe Pyrococcus furiosus was grown on maltose as energy and carbon source. During growth 1 mol maltose was fermented to 3–4 mol acetate, 6–7 mol H₂ and 3–4 mol CO₂. The presence of the following enzyme activities in cell extracts of maltose-grown P. furiosus indicate that the sugar is degraded to pyruvate and H₂ by a modified non-phosphorylated Entner-Doudoroff-pathway (the values given in brackets are specific enzyme activities at 100 °C): Glucose: methyl viologen oxidoreductase (0.03 U/mg); 2-keto-3-deoxy-gluconate aldolase (0.03 U/mg); glyceraldehyde: benzyl viologen oxidoreductase (2.6 U/mg), glycerate kinase (2-phosphoglycerate forming) (0.48 U/mg), enolase (10.4 U/mg), pyruvate kinase (1.4 U/mg). Hexokinase, glucose-6-phosphate dehydrogenase, 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphofructokinase could not be detected. Further conversion of pyruvate to acetate, CO₂ and H₂ involves pyruvate: ferredoxin oxidoreductase (0.4 U/mg; T=60°C with Clostridium pasteurianum ferredoxin as electron acceptor), hydrogen: methyl viologen ixodoreductase (3.4 U/mg) and ADP-dependent acetyl-CoA synthetase (1.9 U/mg). Phosphate acetyl transferase and acetate kinase could not be detected. The ADP-dependent acetyl-CoA synthetase catalyzes ATP synthesis via the mechanism of substrate level phosphorylation and apparently constitutes the only ATP conserving site during maltose catabolism in P. furiosus.This novel pathway of maltose fermentation to acetate, CO₂ and H₂ in the anaerobic archaeon P. furiosus may represent a phylogenetically ancient pathway of sugar fermentation.Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - BV benzyl viologen - CHES cyclohexylamino-ethane sulfonic acid - ABTS 2,2-Azino-di-(3-ethylbenzthiazoliumsulfonate)     

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation,iron release and protein stability

The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase. In this work, a kinetic and spectroscopic investigation on the alkaline chemical denaturation of the protein was performed in an attempt to elucidate the degradation pathway of the iron-sulfur centers in respect to protein unfolding events. For this purpose we investigated cluster dissociation, iron release and protein unfolding by complementary biophysical techniques. We found that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centers are observed. Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether, our data suggest that iron sulfides, which are formed from the release of iron and sulfide resulting from cluster degradation during protein unfolding in alkaline conditions, are in fact responsible for the observed intermediate spectral species, thus disproving the hypothesis suggesting the presence of a linear three-iron center intermediate. Kinetic studies monitored by visible, fluorescence and UV second-derivative spectroscopies have elicited that upon initial perturbation of the tertiary structure the iron-sulfur centers start decomposing and that the presence of EDTA accelerates the process. Also, the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments, further suggesting that the clusters also play a structural role in the maintenance of the conformation of the folded state.     

Stability and fluctuations of amide hydrogen bonds in a bacterial cytochrome c: a molecular dynamics study

Molecular dynamics (MD) simulations on a bacterial cytochrome c were performed to investigate the lifetime and fluctuations of backbone hydrogen bonds and to correlate these data with protection factors for hydrogen exchange measured by NMR spectroscopy (Bartalesi et al. in Biochemistry, 42:10923–10930, 2003). The MD simulations provide a consistent pattern in that long lifetimes of hydrogen bonds go along with small amplitude fluctuations. In agreement with experiments, differences in stability were found with a rather flexible N-terminal segment as compared with a more rigid C-terminal part. Protection factors of backbone hydrogen exchange correlate strongly with the number of contacts but also with hydrogen-bond occupancy, hydrogen-bond survival times, as well as the inverse of fluctuations of backbone atoms and hydrogen-bond lengths derived from MD simulation data. We observed a conformational transition in the C-terminal loop, and significant motion in the N-terminal loop, which can be interpreted as being the structural units involved in the onset of the protein unfolding process in agreement with experimental evidence on mitochondrial cytochrome c. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Gernot Kieseritzky and Giulia Morra both contributed equally to this work.     

Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer

The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.     

Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Effect of a specific inhibitor on the unfolding and refolding kinetics of dimeric triosephosphate isomerase: establishing the dimeric and similarly structured nature of the main transition states on the forward and backward reactions

2-Phosphoglycolate (PGA), a strong competitive inhibitor of the dimeric enzyme triosephosphate isomerase (TIM), brings about a large decrease in the unfolding rate constant of the protein. The data set of rate constants versus ligand concentration may be equally well explained by regarding either a monomeric or a dimeric transition state (TS). However, if the thermodynamics for binding of PGA to native TIM is taken into account, it becomes clear that a dimeric TS is the right assumption. Furthermore, by studying the effect of the ligand on the second-order refolding reaction, we found results indicating similar PGA-binding affinities to be present in the transition states for the rate-limiting steps of the forward and backward reactions. Most likely, therefore, both TS resemble each other in respect to the active site architecture. It should be mentioned, however, that our data do not rule out the possible occurrence of an unstable, (partially) folded monomeric intermediate, which would rapidly interconvert with the unfolded monomer.     

The co-translational folding and interactions of nascent protein chains: a new approach using fluorescence resonance energy transfer

During protein biosynthesis, a nascent protein is exposed to multiple environments and proteins both inside and outside the ribosome that influence nascent chain folding and trafficking. Fluorescence resonance energy transfer between two dyes incorporated into a single nascent chain using aminoacyl-tRNA analogs can directly and selectively monitor changes in nascent chain conformation. This approach recently revealed the existence and functional ramifications of ribosome-mediated folding of nascent membrane proteins inside the ribosome and can be extended to characterize the effects of chaperones and other proteins and ligands on nascent protein folding, interactions, assembly, and avoidance of misfolding and degradation.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Two novel N-terminal coding exons of Prkar1b gene of mouse: identified using a novel approach of in silico and molecular biology techniques

The Prkar1b gene encodes regulatory type 1 beta subunit of protein kinase A. Here we report that mouse R1β gene produces three alternative splice variants (designated as mR1β1, mR1β2 and mR1β3) that have different N-terminal protein structures. New splice variants were identified using combinatorial approach of bioinformatics pipeline involving online available tools and databases, and molecular biology techniques involving RT-PCR, semi-nested PCR and sequencing. Except mR1β3, which was not detected by RT-PCR in brain and muscle tissues of 3day old mice, all three spliced isoforms were found to be ubiquitously expressed in tissues and postnatal developmental stages examined. The presence of different N-termini in isoforms may be important for unique docking interactions with A kinase anchoring proteins.