Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins

The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble.     

The N-terminal part of Binder of SPerm 5 (BSP5), which promotes sperm capacitation in bovine species is intrinsically disordered

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions.     

Microtubule destruction induces tau liberation and its subsequent phosphorylation

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer’s disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation.     

Understanding protein non-folding

This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of a specific 3-D structure. The existence of such proteins does not fit the prevailing structure–function paradigm, which states that a unique 3-D structure is a prerequisite to function. Thus, the protein structure–function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: how were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases?     

Using the concept of Chou's pseudo amino acid composition for risk type prediction of human papillomaviruses

High-risk types of human papillomaviruses (HPVs) are the etiological agents in nearly all cases (99.7%) of cervical cancer, and the HPV E6 protein is one of the two viral oncoproteins which is expressed in virtually all HPV-positive cancers. Therefore, classifying the risk type of HPVs is very useful and necessary for diagnosis and remedy of cervical cancer. To predict and to classify the risk types of HPV by bioinformatics analysis, 96 E6 protein sequences from available databases were obtained. To investigate the risk type of these sequences, PseAAC server, ROC curves and statistical analysis were applied. Our classification was based on some characters of HPV E6 proteins, such as hydrophobicity, hydrophilicity, side chain mass, PK of the α-COOH group, PK of the α-NH3⁺ group and PI at 25 °C. Risk type of 4 unknown HPV types and 25 non-reported HPV types were also predicted. These results show that bioinformatics based theoretical approaches can direct and simplify experimental studies.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus     

蛋白质的选择性降解机制——2004年诺贝尔化学奖部分工作介绍

如何识别和选择性降解蛋白质是细胞生命过程中的重要环节.泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式.对于需要清除的蛋白质,通过其赖氨酸残基侧链ε-氨基连接多聚泛素链（降解标签）,继而在蛋白酶体中被降解.这种选择性降解机制对于维持蛋白质在细胞内含量的动态平衡起到了关键性作用.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.