Refinement of protein termini in template-based modeling using conformational space annealing

The rapid increase in the number of experimentally determined protein structures in recent years enables us to obtain more reliable protein tertiary structure models than ever by template-based modeling. However, refinement of template-based models beyond the limit available from the best templates is still needed for understanding protein function in atomic detail. In this work, we develop a new method for protein terminus modeling that can be applied to refinement of models with unreliable terminus structures. The energy function for terminus modeling consists of both physics-based and knowledge-based potential terms with carefully optimized relative weights. Effective sampling of both the framework and terminus is performed using the conformational space annealing technique. This method has been tested on a set of termini derived from a nonredundant structure database and two sets of termini from the CASP8 targets. The performance of the terminus modeling method is significantly improved over our previous method that does not employ terminus refinement. It is also comparable or superior to the best server methods tested in CASP8. The success of the current approach suggests that similar strategy may be applied to other types of refinement problems such as loop modeling or secondary structure rearrangement.     

Participation of protein sequence termini in crystal contacts

The analysis of the crystal packing interactions, in a nonredundant set of high resolution and monomeric globular protein crystal structures, shows that the residues located at the N- and C-termini of the sequence tend to participate in packing interaction more often than expected and that often they interact with each other. Since the sequence termini are, in general, conformationally very flexible and since they host electrical charges of opposite sign, it can be hypothesized that they play a crucial role in the early formation of the nonphysiological contacts that bring to protein crystallization. It is thus not surprising that modest lengthening/shortening of the sequence termini have often a dramatic effect on protein crystallogenesis.     

A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity

Metabotropic glutamate receptor subtype 1a (mGluR1a) associates with the proteins mediating its receptor activity, suggesting a complex-controlled function of mGluR1a. Here, using glutathione-S-transferase pull-down, co-immnoprecipitation and immnoflurescence assays in vitro and in vivo, we have found CFTR-associated ligand (CAL) to be a novel binding partner of mGluR1a, through its PSD95/discslarge/ZO1homology domain. Deletion of mGluR1a-carboxyl terminus (CT) or mutation of Leu to Ala in the CT of mGluR1a reduces the association, indicating the essential binding region of mGluR1a for CAL. Functionally, the interaction of mGluR1a with CAL was shown to inhibit mGluR1a-mediated ERK1/2 activation, without an apparent effect, via the C-terminal-truncated receptor. These findings might provide a novel mechanism for the regulation of mGluR1a-mediated signaling through the interaction with CAL.

Structured summary

MINT-6797987, MINT-6798009:

NHERF-2 (uniprotkb:Q15599) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by proteinarray (MI:0089)

MINT-6798026, MINT-6798048, MINT-6798066:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by pull down (MI:0096)

MINT-6797953, MINT-6797970:

NHERF-1 (uniprotkb:O14745) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6797935:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6798084:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by filter binding (MI:0049)

MINT-6798134:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by anti tag coimmunoprecipitation (MI:0007)

MINT-6798158:

CAL (uniprotkb:B4F775) physically interacts (MI:0218) with mGluR1a (uniprotkb:Q9R0W0) by anti bait coimmunoprecipitation (MI:0006)

MINT-6798233:

CAL (uniprotkb:Q9HD26) colocalizes (MI:0403) with mGluR1a (uniprotkb:Q9R0W0) by fluorescence microscopy (MI:0416)

     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

The in vivo and in vitro aggregation properties of globular proteins correlate with their conformational stability: the SH3 case

Protein misfolding and deposition underlie an increasing number of debilitating human disorders and constitute a problem of major concern in biotechnology. In the last years, in vitro studies have provided valuable insights into the physicochemical principles underlying protein aggregation. Nevertheless, information about the determinants of protein deposition within the cell is scarce and only a few systematic studies comparing in vitro and in vivo data have been reported. Here, we have used the SH3 domain of α-spectrin as a model globular protein in an attempt to understand the relationship between protein aggregation in the test-tube and in the more complex cellular environment. The investigation of the aggregation in Escherichia coli of this domain and a large set of mutants, together with the analysis of their sequential and conformational properties allowed us to evaluate the contribution of different polypeptidic factors to the cellular deposition of globular proteins. The data presented here suggest that the rules that govern in vitro protein aggregation are also valid in in vivo contexts. They also provide relevant insights into intracellular protein deposition in both conformational diseases and recombinant protein production.     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

Evolutionary conserved N-terminal region of human muscle fructose 1,6-bisphosphatase regulates its activity and the interaction with aldolase

N-terminal residues of muscle fructose 1,6-bisphosphatase (FBPase) are highly conserved among vertebrates. In this article, we present evidence that the conservation is responsible for the unique properties of the muscle FBPase isozyme: high sensitivity to AMP and Ca(2+) inhibition and the high affinity to muscle aldolase, which is a factor desensitizing muscle FBPase toward AMP and Ca(2+). The first N-terminal residue affecting the affinity of muscle FBPase to aldolase is arginine 3. On the other hand, the first residue significantly influencing the kinetics of muscle FBPase is proline 5. Truncation from 5-7 N-terminal residues of the enzyme not only decreases its affinity to aldolase but also reduces its k-(cat) and activation by Mg(2+), and desensitizes FBPase to inhibition by AMP and calcium ions. Deletion of the first 10 amino acids of muscle FBPase abolishes cooperativity of Mg(2+) activation and results in biphasic inhibition of the enzyme by AMP. Moreover, this truncation lowers affinity of muscle FBPase to aldolase about 14 times, making it resemble the liver isozyme. We suggest that the existence of highly AMP-sensitive muscle-like FBPase, activity of which is regulated by metabolite-dependent interaction with aldolase enables the precise regulation of muscle energy expenditures and might contributed to the evolutionary success of vertebrates.     

N-terminal extension changes the folding mechanism of the FK506-binding protein

Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.     

Human interferon gamma: significance of the C-terminal flexible domain for its biological activity

The significance of the C-terminal part of human interferon gamma (hIFNgamma) for its biological activity was studied by 3(')-end gene mutagenesis. A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus. To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNgamma proteins was measured in clear cell lysates containing the soluble fractions only. The deletion of the C terminus had a two-step effect on both hIFNgamma antiviral and antiproliferative activities. Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNgamma, the deletion of more than 9 amino acids had an opposite effect. The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNgamma. The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the alpha-helical domain F.