Local cultivars of pearl millet in Saudi Arabia are known to tolerate extreme heat and drought stress. In the current study, the sequences of internal-transcribed spacers (ITSs) of six pearl millet cultivars were sequenced and analysed to investigate the genetic diversity among the local cultivars. The nucleotide polymorphism, secondary structures and phylogenetics were analysed for ITS sequences of the six local cultivars. The obtained sequences were 772–774 base pairs (bp) in length, including complete sequences of the ITS1–5.8S–ITS2 region and partial sequences of 18S and 26S rRNA. The nucleotide diversity among cultivars was higher in ITS2 sequences than ITS1 sequences. The ITS2 had four variable nucleotide sites in three native cultivars, whereas the ITS1 contained one base insertion. The secondary structures of the ITS1 and 5.8S region were highly conserved among the six cultivars and contained some motifs that are conserved across Viridiplantae. However, the ITS2 secondary structure for the two native cultivars, Sayah and Jazan, was distinct from the other cultivars, which confirms the applicability of the ITS2 sequence in distinguishing between genetically close taxa. Additionally, the phylogenetic analysis of the six investigated cultivars and 31 pearl millet accessions from the NCBI database showed close relationships between the local accessions and NCBI accessions from India and France. However, the local cultivar Sayah appeared to be distinct from the other cultivars in the phylogenetic trees. This study provides insights into the polymorphism within local pearl millet cultivars which is important for the identification and conservation of these cultivars.

As part of our ongoing research on Peltigera, we recognize a morphologically and phylogenetically distinct new species, Peltigera shennongjiana L. F. Han & S. Y. Guo, from the Shennongjia region of Central China. It is distinguished from other members of the P. canina-group by the presence of abundant phyllidia and flat, branched lobules along the margin or laminal cracks, short lobes, and a pruinose, usually greyish upper surface. The various populations sampled share identical ITS nr DNA sequences, of which the ITS2 regions are characterized by a unique secondary structure. Furthermore, we provide a detailed comparison of the characteristics of P. shennongjiana with morphologically similar species and a key to Peltigera species reported from China.

Protein structure influences the accessibility of enzymes for digestion. The proportion of intramolecular β-sheets in the secondary structure of native proteins has been related to a decrease in protein digestibility. Changes to proteins that can be considered positive (for example, denaturation and random coil formation) or negative (for example, aggregation and Maillard reactions) for protein digestibility can occur simultaneously during processing. The final result of these changes on digestibility seems to be a counterbalance of the occurrence of each phenomenon. Occurrence of each phenomenon depends on the conditions applied, but also on the source and type of the protein that is processed. The correlation between denaturation enthalpy after processing and protein digestibility seems to be dependent on the protein source. Heat seems to be the processing parameter with the largest influence on changes in the structure of proteins. The effect of moisture is usually limited to the simultaneous application of heat, but increasing level of moisture during processing usually increases structural changes in proteins. The effect of shear on protein structure is commonly studied using extrusion, although the multifactorial essence of this technology does not allow disentanglement of the separate effects of each processing parameter (for example, heat, shear, moisture). Although most of the available literature on the processing of feed ingredients reports effects on protein digestibility, the mechanisms that explain these effects are usually lacking. Clarifying these mechanisms could aid in the prediction of the nutritional consequences of processing conditions.

In a manner similar to many other groups of organisms, the tapeworm order Proteocephalidea poses a difficult phylogenetic problem if treated on the basis of single-gene analysis. Since the biogeography and host distribution of proteocephalideans make these tapeworms a potentially interesting model for evolutionary and co-evolutionary studies, we tried to resolve their phylogenetic relationships by applying a multi-gene approach. The ITS2 sequences and V4 hypervariable loop of 18S rRNA were obtained for 43 and 35 proteocephalidean taxa, respectively, and combined with other sequences available in the GenBank. The phylogenetic analysis of the combined DNA set was confronted with characters derived from ITS2 secondary structures. Using this approach, a species-rich Neotropical lineage of proteocephalideans could be reliably resolved. The phylogenetic relationships within this group show a high degree of phylogeny-independent host distribution. The reconstruction of ITS2 secondary structure revealed a universal 4-domain arrangement, which is conserved across a wide range of Neodermata. Several motifs of the secondary structure could be mapped to the phylogenetic tree as possible clade synapomorphies.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5′ domain of BC1 RNA, derived from tRNAAla, does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNAAla domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3′ end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl- and lipoyl-dependent enzymes is post-translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation. Counterparts of the E. coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported. Computational analysis based on PSI-BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module. Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X-ray crystal structure of the E. coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes.

We describe a new classifier for protein secondary structure prediction that is formed by cascading together different types of classifiers using neural networks and linear discrimination. The new classifier achieves an accuracy of 76.7% (assessed by a rigorous full Jack-knife procedure) on a new nonredundant dataset of 496 nonhomologous sequences (obtained from G.J. Barton and J.A. Cuff). This database was especially designed to train and test protein secondary structure prediction methods, and it uses a more stringent definition of homologous sequence than in previous studies. We show that it is possible to design classifiers that can highly discriminate the three classes (H, E, C) with an accuracy of up to 78% for β-strands, using only a local window and resampling techniques. This indicates that the importance of long-range interactions for the prediction of β-strands has been probably previously overestimated.

The amino terminal domain of enzyme I (residues 1–258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, ΔH ≅ 140 ± 10 (160) kcal mol−1 and ΔCp ≅ 2.7 (3.3) kcal K−1 mol−1. Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) °C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO32− due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ-carboxyl of Glu189 and the ε-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K′A = 3 ± 1 × 105 M−1 for EIN and ∼1.2 × 105 M−1 for EI.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3JHNHA coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and β-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

A search for the occurrence of the rare π-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the π-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight π-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the π-helix has evolved to provide unique structural features within a variety of proteins.

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.

The assumption that the intrinsic α-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by ∼0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (±0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (±0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized α-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.

Group I introns constitute excellent systems for analyzing the relationship between RNA tertiary folding and catalysis. Within a hierarchical framework interpretation of RNA folding, secondary structure motifs subtend RNA three-dimensional (3D) architecture. Thus, mutations in two-dimensional motifs are expected to have effects different from those disrupting 3D contacts. Using UV spectroscopy, we have studied the influence of nucleotide substitutions, in both secondary and tertiary structure elements, on the thermal stability of the tertiary folding of the bacteriophage T4 td group I intron. Further, we present a quantitative analysis of the relationship between the splicing efficiency in vivo and the stability of the intron structure as monitored by UV melting curves. We conclude that the stability of the tertiary structure of a group I intron as measured by UV melting is generally a good indication of its ability to splice in vivo.

NMR offers the possibility of accurate secondary structure for proteins that would be too large for structure determination. In the absence of an X-ray crystal structure, this information should be useful as an adjunct to protein fold recognition methods based on low resolution force fields. The value of this information has been tested by adding varying amounts of artificial secondary structure data and threading a sequence through a library of candidate folds. Using a literature test set, the threading method alone has only a one-third chance of producing a correct answer among the top ten guesses. With realistic secondary structure information, one can expect a 60–80% chance of finding a homologous structure. The method has then been applied to examples with published estimates of secondary structure. This implementation is completely independent of sequence homology, and sequences are optimally aligned to candidate structures with gaps and insertions allowed. Unlike work using predicted secondary structure, we test the effect of differing amounts of relatively reliable data.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine α-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: α-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.