On the mechanism of protein fold‐switching by a molecular sensor

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild‐type fold (N) to a circularly‐permuted fold (N′), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order–disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D_9k. A simple three‐state model for fold switching holds that N and N′ interconvert through the unfolded state. This model predicts that the fundamental properties of the switch—calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate—can be controlled by altering the relative stabilities of N and N′. We find that selectively destabilizing N or N′ changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole‐molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Detection and Partial Characterization of Milk vetch dwarf virus Isolates from Faba Bean (Vicia faba L.) in Yunnan Province, China

A virus disease of faba bean ( Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus . The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.     

MGMT Ile143Val polymorphism,dietary factors and the risk of breast,colorectal and prostate cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study

O⁶-Methylguanine-DNA methyltransferase (MGMT) repairs DNA damage caused by alkylating agents including N-nitroso compounds from diet. MGMT Ile143Val polymorphism may lead to less DNA damage repair and increased cancer risk depending on the environmental exposures. We investigated interactions between dietary factors and the MGMT Ile143Val polymorphism in relation to breast, colorectal and prostate cancer risk. There were 276/1498, 273/2984 and 312/1486 cases/controls for the breast, colorectal and prostate cancer studies respectively; all nested within the EPIC-Norfolk study, a prospective cohort of approximately 25,000 men and women aged 40–79. Baseline 7-day food diary data were collected for dietary assessment. MGMT Ile143Val polymorphism was not overall associated with breast, colorectal and prostate cancer risk. There was a significant interaction between this polymorphism and intake of red and processed meat on colorectal cancer risk (P_interaction = 0.04) suggesting an increased risk among carriers of the variant genotype compared to the MGMT Ile143Ile common genotype. A lower colorectal cancer risk was seen with higher intake of vitamin E and carotene among the variant genotype group but not in the common genotype group (P_interaction = 0.009 and P_interaction = 0.005 for vitamin E and carotene, respectively). A higher prostate cancer risk was seen with higher alcohol intake among the variant genotype (OR = 2.08, 95% CI = 1.21–3.57, P_interaction = 0.0009) compared to the common genotype with lower alcohol intake. In this UK population, the MGMT Ile143Val polymorphism was not overall associated with breast, colorectal and prostate cancer risk. There was evidence for this polymorphism playing a role in modulating the risk of prostate cancer in presence of alcohol. For colorectal cancer, the MGMT Ile143Val polymorphism may confer increased or decreased risk depending on the dietary exposure.     

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.     

Mutation of the hydrophobic motif in a phosphorylation-deficient mutant renders protein kinase C delta more apoptotically active

Protein kinase C delta (PKCδ) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCδ is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCδ. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCδ activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCδ (PKCδ-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCδ apoptotically more active. Furthermore, we found that the mutant PKCδ-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart.     

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.     

Development of multidrug resistance type I Cmdr1 expression in chicken embryonic gonads

A primary role of P-glycoprotein (P-gp), encoded by the multidrug resistance type I gene, is to protect against naturally occurring xenotoxics. Recently, the preferential expression of chicken multidrug resistance type I (Cmdr1) was identified in the embryonic gonads during the early periods of development. Here we investigated the expression of Cmdr1 and P-gp in the gonads during embryogenesis, and compared to that in the ovarian follicles of domestic hens (Gallus gallus). As revealed by immunohistochemistry, P-gp was highly expressed in theca cells of mature follicles, whereas the expression was low in immature follicles. Immunohistochemical analysis showed that expression of Cmdr1-type P-gp was very low in embryonic gonads. Cmdr1 mRNA was undetectable in the gonads of 5-day embryos (E5) by RT-PCR, whereas Cmdr1 mRNA was significantly detectable in the developing gonads at E9 and E21. In the testicular tissues, germ cells were distributed along developing seminiferous cords as identified by a specific marker gene, whereas Cmdr1-type P-gp positive cells were observed evenly on testicular tissues. Collectively, it is concluded that Cmdr1 expression is initiated in the chicken ovary and testis after sexual differentiation, but expression of Cmdr1-type P-gp is very low through embryogenesis.     

Genetic constraints on protein evolution

Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.