Length-dependent prediction of protein intrinsic disorder

Background  

Due to the functional importance of intrinsically disordered proteins or protein regions, prediction of intrinsic protein disorder from amino acid sequence has become an area of active research as witnessed in the 6th experiment on Critical Assessment of Techniques for Protein Structure Prediction (CASP6). Since the initial work by Romero et al. (Identifying disordered regions in proteins from amino acid sequences, IEEE Int. Conf. Neural Netw., 1997), our group has developed several predictors optimized for long disordered regions (>30 residues) with prediction accuracy exceeding 85%. However, these predictors are less successful on short disordered regions (≤30 residues). A probable cause is a length-dependent amino acid compositions and sequence properties of disordered regions.     

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.     

Identification of intrinsic order and disorder in the DNA repair protein XPA

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single-stranded DNA binding protein

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling.     

Minor fibrillar collagens,variable regions alternative splicing,intrinsic disorder,and tyrosine sulfation

Minor fibrillar collagen types Ⅴ and Ⅺ,are those less abundant than the fibrillar collagen types Ⅰ,Ⅱ and Ⅲ.The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region.Genomic variation and,in some cases,extensive alternative splicing contribute to the unique sequence characteristics of the variable region.While unique expression patterns in tissues exist,the functions and biological relevance of the variable regions have not been elucidated.In this review,we summarize the existing knowledge about expression patterns and biological functions of the collagen types Ⅴ and Ⅺ alpha chains.Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggests the potential for a shared function.The alternative splicing,conservation of biochemical characteristics in light of low sequence conservation,and evidence for intrinsic disorder,suggest modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.     

Minor fibrillar collagens,variable regions alternative splicing,intrinsic disorder,and tyrosine sulfation

Minor fibrillar collagen types V and XI, are those less abundant than the fibrillar collagen types I, II and III. The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region. Genomic variation and, in some cases, extensive alternative splicing contribute to the unique sequence characteristics of the variable region. While unique expression patterns in tissues exist, the functions and biological relevance of the variable regions have not been elucidated. In this review, we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and XI alpha chains. Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggests the potential for a shared function. The alternative splicing, conservation of biochemical characteristics in light of low sequence conservation, and evidence for intrinsic disorder, suggest modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.     

Domain structure and DNA binding regions of beta protein from bacteriophage lambda

beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.     

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D'-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D'-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.     

DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.     

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 598–606.     

细菌RNA结合蛋白Hfq的DNA压缩和复制作用研究进展

RNA结合蛋白(RNA-Binding Protein)Hfq是一种重要的细菌转录后调节因子,之前对Hfq的研究大多集中在该蛋白对小分子非编码RNA(Small Non-Coding RNA,sRNA)和mRNA的作用上.Hfq最典型的功能是促进sRNA与其靶标mRNA碱基配对,在转录后介导对RNA的稳定性和翻译的调控...     

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.     

Autogenous translation regulation by Escherichia coli ATPase SecA may be mediated by an intrinsic RNA helicase activity of this protein.

The seven conserved motifs typical of the helicase superfamily II have been identified in the sequences of Escherichia coli protein SecA, an ATPase mediating protein translocation across the inner membrane of the bacterium, and its Bacillus subtilis homolog Div. It is hypothesized that SecA and Div possess an RNA helicase activity and may couple ATP hydrolysis both to membrane translocation of proteins, and to hairpin unwinding in their own mRNAs, leading to the known autogenous regulation of translation.     

Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔH^cal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.