Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module

An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.     

Prediction of the initial folding sites and the entire folding processes for Ig-like beta-sandwich proteins

Describing the whole story of protein folding is currently the main enigmatic problem in molecular bioinformatics study. Protein folding mechanisms have been intensively investigated with experimental as well as simulation techniques. Since a protein folds into its specific 3D structure from a unique amino acid sequence, it is interesting to extract as much information as possible from the amino acid sequence of a protein. Analyses based on inter-residue average distance statistics and a coarse-grained Gō-model simulation were conducted on Ig and FN3 domains of a titin protein to decode the folding mechanisms from their sequence data and native structure data, respectively. The central region of all domains was predicted to be an initial folding unit, that is, stable in an early state of folding. This common feature coincides well with the experimental results and underscores the significance of the β-sandwich proteins' common structure, namely, the key strands for folding and the Greek-key motif, which is located in the central region. We confirmed that our sequence-based techniques were able to predict the initial folding event just next to the denatured state and that a 3D-based Gō-model simulation can be used to investigate the whole process of protein folding.     

Folding superfunnel to describe cooperative folding of interacting proteins

This paper proposes a generalization of the well‐known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein–protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein–protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009–1016. © 2016 Wiley Periodicals, Inc.     

Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework.

We have identified three major blocks of amino acid homology shared by the transit peptides of two nuclear-encoded chloroplast proteins, the light-harvesting chlorophyll a/b-protein (LHCP) II of the thylakoid membrane and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of the stroma. These previously unrecognized homology blocks lie at the beginning, middle and end of both transit sequences, and are separated by differing lengths of unshared (interblock) sequence in the two proteins. These interblocks may be dispensible or they might confer a specific property on the individual proteins, such as facilitating proper compartmentalization within the chloroplast. We propose that these three shared sequence elements form a common framework in transit-bearing chloroplast precursors which mediates the common functions performed by each transit peptide. Ferredoxin, the only other such nuclear-encoded protein for which a published transit sequence exists, conforms to the predictions of this hypothesis. These findings stand in contrast to mitochondrial leader sequences and the well-studied signal peptides of secretory and certain integral membrane proteins in which no such framework has been observed.     

Antigenic modulation and down regulation-their similarities suggest they may have a common origin

Antigenic modulation, the antibody-induced disappearance of membrane-associated determinants of neoplastic cells, has a fundamental similarity to the down regulation of receptors for hormones and other ligands affecting the physiology of various nonneoplastic cell types. After hormone stimulation, cells previously responsive become refractory to further stimulation. Diminution in responsiveness correlates with a quantitative reduction in the numbers of specific complementary hormone receptors present. Murine leukemia cells, as one example "escape" antibody-mediated destruction through the mechanism of antigenic modulation. Resistance of the cells correlates with a reduction of the numbers of specific antigens present. In both down regulation and modulation, continued cellular metabolism in the absence of ligand leads to a re-expression of the hormone receptor/membrane antigen involved. It is postulated that the two phenomena have evolved from similar underlying cellular mechanisms.     

Folding barrier in horse cytochrome c: support for a classical folding pathway

Native-state structures and conformations of ferrocytochrome c, nitrosylcytochrome c, and carbonmonoxycytochrome c are very similar. They are, however, immensely different from each other in terms of thermodynamic stability. The dramatic destabilization of ferrocytochrome c to the extent of 12 kcal mol(-1) produces no effect on the folding rate, and this is so in spite of the fact that all three test-tube variants fold in an apparent two-state manner. For all three proteins the folding barrier is early in time, sizable in energy, and is of the same magnitude (approximately 6.5 kcal mol(-1)). These results raise some challenges to the "new view" of protein folding. An early transition state, the search for which consumes most of the observed folding time, is suggested.     

Sec-dependent pathway and DeltapH-dependent pathway do not share a common translocation pore in thylakoidal protein transport.

Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the DeltapH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the DeltapH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the DeltapH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the DeltapH-dependent pathway.     

Seed storage proteins of spermatophytes share a common ancestor with desiccation proteins of fungi

The legumin- and vicilin-like seed storage globulins of spermatophytes are specifically accumulated during embryogenesis and seed development. Previous studies have shown that a precursor common to both legumin and vicilin genes might have evolved by duplication from a single-domain ancestral gene. We here report that amino acid sequences of legumin and vicilin domains share statistically significant similarity to the germination-specific germins of wheat as well as to the spherulation-specific spherulins of myxomycetes. This conclusion is further supported by the derived intron-exon structure of a spherulin gene. Spherulins are thought to be involved in tissue desiccation or hydration. It is suggested that the present-day seed globulins of spermatophytes have evolved from a group of ancient proteins functional in cellular desiccation/hydration processes. Correspondence to: H. Bäumlein     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Biochemical properties of phosphatidylcholine-binding proteins that share common antigenic determinants with Fc gamma 2b receptor

Biochemical and immunological properties of biosynthetically radiolabeled phosphatidylcholine-(PC-) binding proteins were investigated. The PC-binding proteins were extracted from the detergent lysate of biosynthetically radiolabeled P388D1 cells by affinity chromatography on PC-Sepharose and filtered through a Sephadex G-100 gel column in the presence of 6 M urea. Isoelectric focusing of the gel-filtered materials in the presence of 6 M urea revealed the presence of a major protein component of pIe of 5.8 and minor heterogeneous cellular proteins. The yield of the electrofocused PC-binding proteins based on protein determination by Lowry's method ranged from 0.7 to 4 mg per 10(9) cells. The purified PC-binding proteins appeared to be tightly associated with Triton X-100 and phospholipids in the weight ratio of 0.57 and 0.05 g/g of proteins, respectively. The majority of lipids that could be extracted from the PC-binding proteins by chloroform/methanol (2:1 v/v) are free fatty acids, whereas lipids extracted from Pronase-treated PC-binding proteins contained phosphatidylethanolamine. By amino acid analysis, the purified PC-binding proteins were found to consist of a minimum of 417 amino acid residues, suggesting a minimum molecular weight of about 38 000 for this protein. Results of radiolabeling experiments with [3H]glucosamine and amino acid analysis both showed the presence of a mole of glucosamine per a mole of the PC-binding proteins, suggesting their glycoprotein nature. About 40% of the purified PC-binding proteins coprecipitated with monoclonal anti-Fc gamma 2bR antibody (2.4G2) in detergent-containing buffer, whereas only 6% of the isolated IgG binding proteins reacted with this antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ameloblastin and amelogenin share a common secretory pathway and are co-secreted during enamel formation.

The epithelially-derived ameloblasts secrete two main categories of extracellular matrix proteins, amelogenins (AMEL) and nonamelogenins. These proteins assume differential distributions in the forming enamel layer and thereby regulate deposition and structuring of the mineral phase. The objective of this study was to elucidate whether their distribution results from distinctive physicochemical behaviors or differences in intracellular routing. Dual-immunogold labeling was used to visualize the presence of AMEL and ameloblastin (AMBN), the major nonamelogenin, and quantify the proportion of secretory granules containing one or both of these proteins in ameloblasts during the phase of appositional growth of the enamel layer in continuously-erupting rat incisors. Some rats were treated with brefeldin A (BFA) to generate a synchronized cohort of newly-formed secretory granules. The results show that nearly 70% of granules contain both AMEL and AMBN, 13% label only for AMBN and 1% only for AMEL. These proportions reach 98% (AMEL+AMBN) and 2% (AMBN only) following BFA treatment. The observation that AMEL is almost always packaged with AMBN suggests a functional association between these two proteins. The subpopulation of granules containing only AMBN could be responsible for augmenting its local concentration along secretory surfaces against which hydroxyapatite crystals actively elongate.     

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.     

Folding studies of two hydrostatic pressure sensitive proteins

High hydrostatic pressure combined with various spectroscopies is a powerful technique to study protein folding. An ideal model system for protein folding studies should have the following characteristics. (1) The protein should be sensitive to pressure, so that the protein can be unfolded under mild pressure. (2) The folding process of the protein should be easily modulated by several chemical or physical factors. (3) The folding process should be easily monitored by some spectroscopic parameters. Here, we summarized the pressure induced folding studies of two proteins isolated from spinach photosystem II, namely the 23-kDa and the 33-kDa protein. They have all the characteristics mention above and might be an ideal model protein system for pressure studies.     

mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation.

During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.     

CD studies on films of amyloid proteins and polypeptides: quantitative g-factor analysis indicates a common folding motif

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a "cross-beta" structure, with extended beta-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P. McPhie, Analytical Biochemistry, 2001, Vol. 293, pp. 109-119). Amyloid films of Abeta-(1-40) peptide, beta-2-microglobulin, insulin, and three homopolypeptides show very intense CD spectra, compatible with the presence of a beta-helix-like structure, arranged in a common framework in the fibrils. The extent of this structure was estimated as 45-80% in the protein fibrils and 30-80% in the polypeptide fibrils.     

Contact potential that recognizes the correct folding of globular proteins.

We have devised a continuous function of interresidue contacts in globular proteins such that the X-ray crystal structure has a lower function value than that of thousands of protein-like alternative conformations. Although we fit the adjustable parameters of the potential using only 10,000 alternative structures for a selected training set of 37 proteins, a grand total of 530,000 constraints was satisfied, derived from 73 proteins and their numerous alternative conformations. In every case where the native conformation is adequately globular and compact, according to objective criteria we have developed, the potential function always favors the native over all alternatives by a substantial margin. This is true even for an additional three proteins never used in any way in the fitting procedure. Conformations differing only slightly from the native, such as those coming from crystal structures of the same protein complexed with different ligands or from crystal structures of point mutants, have function values very similar to the native's and always less than those of alternatives derived from substantially different crystal structures. This holds for all 95 structures that are homologous to one or another of various proteins we used. Realizing that this potential should be useful for modeling the conformation of new protein sequences from the body of protein crystal structures, we suggest a test for deciding whether a nearly correct approximation to the native conformation has been found.     

Factors that affect the folding ability of proteins

The folding ability of a heteropolymer model for proteins subject to Monte Carlo dynamics on a simple cubic lattice is shown to be strongly correlated with the stability of the native state. We consider a number of estimates of the stability that can be determined without simulation, including the energy gap between the native state and the structurally dissimilar part of the spectrum (Z score) and, for sequences with fully compact native states, the gap in energy between the native and first excited fully compact states. These estimates are found to be more robust predictors of folding ability than a parameter sigma that requires simulation for its evaluation: sigma = 1 - Tf/Ttheta, where Tf is the temperature at which the fluctuation of an order parameter is at its maximum and Ttheta is the temperature at which the specific heat is at its maximum. We show that the interpretation of Ttheta as the collapse transition temperature is not correct in general and that the correlation between sigma and the folding ability arises from the fact that sigma is related to the energy gap (Z score).     

Computer-based redesign of a protein folding pathway.

A fundamental test of our current understanding of protein folding is to rationally redesign protein folding pathways. We use a computer-based design strategy to switch the folding pathway of protein G, which normally involves formation of the second, but not the first, beta-turn at the rate limiting step in folding. Backbone conformations and amino acid sequences that maximize the interaction density in the first beta-hairpin were identified, and two variants containing 11 amino acid replacements were found to be approximately 4 kcal mol-1 more stable than wild type protein G. Kinetic studies show that the redesigned proteins fold approximately 100 x faster than wild type protein and that the first beta-turn is formed and the second disrupted at the rate limiting step in folding.