Molecular Dynamics Study on the Effects of Charged Amino Acid Distribution Under low pH Condition to the Unfolding of Hen Egg White Lysozyme and Formation of Beta Strands.

Aggregation of unfolded or misfolded proteins into amyloid fibrils can cause various diseases in humans. However, the fibrils synthesized in vitro can be developed toward useful biomaterials under some physicochemical conditions. In this study, atomistic molecular dynamics simulations were performed to address the mechanism of beta-sheet formation of the unfolded hen egg white lysozyme (HEWL) under a high temperature and low pH. Simulations of the protonated HEWL at pH 2 and the non-protonated HEWL at pH 7 were performed at the highly elevated temperature of 450 K to accelerate the unfolding, followed by the 333 K temperature in some previous in vitro studies. The simulations showed that HEWL unfolded faster and refolded into structures with higher beta-strand contents at pH 2. The mechanism of beta-strand formation at the earlier stage of amyloidosis was addressed in terms of the radial distribution of amino acids, affected by the high protonation level at a low pH.

Fragile protein folds: Sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations

Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal beta-strand. Using this protein as a test case, we advance the concept of a fragile fold, a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note, we find that factors which impact the HI loop preceding the shifted I(beta)-strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wildtype conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.

High-Pressure NMR Reveals Volume and Compressibility Differences Between Two Stably Folded Protein Conformations

ABSTRACTProteins often interconvert between different conformations in ways critical to their function. While manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic beta-strand slip as it switches between two stably-folded conformations. Coupling high pressure and biomolecular NMR, we obtained the first quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wildtype equivalent, and the interconversion proceeds through a chiefly-unfolded intermediate state. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

The polymorphisms of NR5A1 gene in azoospermic men in Sichuan, China

Background: Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator involved in the hypothalamic-pituitary-steroidogenic organ development. Recently, heterozygous mutations in NR5A1 were found may contribute to the male infertility aetiology. Here, we investigated the association of polymorphisms in NR5A1 gene with azoospermic men in Sichuan, China. Methods: We have performed the NR5A1 gene direct secquencing in a cohort of 102 well-characterised idiopathic Chinese azoospermic infertile men versus 103 fertile men, who were selected by Semen analysis, Karyotype analysis and Y-chromosomal AZF deletion screening. We identified two previously described missense p. Results: Gly146Ala (rs1110061; c.437 G>C) and p.Arg313His (c.938G>A), and the frequency of 437C ( [OR] 1.846, 95% [CI] 1.227-2.778, P=0.003), 437GC (OR =1.884 , 95% CI =1.037-3.422 , P =0.037 ) and 437CC (OR =3.586 , 95% CI =1.397-9.206 , P =0.006 ) were found to be increased significantly in azoospermic patients while no mutations in control .Moreover, one novel heterozygous p.Ser322ILe (c.965 G >A) missense mutation was found in 8 patients which highly conserved serine to isoleucine shown in the Beta strand domain on SF-1 protein. Conclusions: This is the first study, according to our knowledge, to investigate the association between the polymorphisms of NR5A1 gene and azoospermic men in China, and these results suggest that the Gly146Ala polymorphism may be a susceptibility factor for the azoospermic men in Sichuan, China.

Invertebrate Metaxins 1 and 2: Widely Distributed Proteins Homologous to Vertebrate Metaxins Implicated in Protein Import into Mitochondria

ABSTRACTMetaxin 1 and 2 genes, previously investigated in vertebrates, are shown to be widely distributed among invertebrates. But metaxin 3 is absent. The predicted proteins of the invertebrate metaxins were initially identified by homology with human metaxin 1 and 2 proteins, and by the presence of characteristic GST_Metaxin protein domains. Invertebrate metaxins were revealed for a variety of phyla, including Echinodermata, Cnidaria, Porifera, Chordata, Arthropoda, Mollusca, Brachiopoda, Placozoa, and Nematoda. Metaxins were also found in insects (Arthropoda) of different taxonomic orders: Diptera, Coleoptera, Lepidoptera, Hymenoptera, and Blattodea. Invertebrate and human metaxin 1 proteins have about 41% identical amino acids, while metaxin 2 proteins have about 49% identities. Invertebrate and vertebrate metaxins share the same characteristic protein domains, further strengthening the identification of the invertebrate proteins as metaxins. The domains are, for metaxin 1, GST_N_Metaxin1_like, GST_C_Metaxin1_3, and Tom37. For metaxin 2, they are GST_N_Metaxin2, GST_C_Metaxin2, and Tom37. Phylogenetic trees show that invertebrate metaxin 1 and metaxin 2 proteins are related, but form separate groups. The invertebrate proteins are also closely related to vertebrate metaxins, though forming separate clusters. These phylogenetic results indicate that all metaxins likely arose from a common ancestral sequence. The neighboring genes of the invertebrate metaxin 1 and 2 genes are largely different for different invertebrate species. This is unlike the situation with vertebrate metaxin genes, where, for example, the metaxin 1 gene is adjacent to the thrombospondin 3 gene. The dominant secondary structures predicted for the invertebrate metaxins are alpha-helical segments, with little beta-strand. The conserved pattern of helical segments is the same as that found for vertebrate metaxins 1, 2, and 3.