Radius of gyration as an indicator of protein structure compactness

Identification and study of the main principles underlying the kinetics and thermodynamics of protein folding generate a new insight into the factors that control this process. Statistical analysis of the radius of gyration for 3769 protein domains of four major classes (α, β, α/β, and α + β) showed that each class has a characteristic radius of gyration that determines the protein structure compactness. For instance, α proteins have the highest radius of gyration throughout the protein size range considered, suggesting a less tight packing as compared with β-and (α + β)-proteins. The lowest radius of gyration and, accordingly, the tightest packing are characteristic of α/β-proteins. The protein radius of gyration normalized by the radius of gyration of a ball with the same volume is independent of the protein size, in contrast to compactness and the number of contacts per residue.     

Radius of gyration of plasmid DNA isoforms from static light scattering

Despite the extensive interest in applications of plasmid DNA, there have been few direct measurements of the root mean square radius of gyration, R_G, of different plasmid isoforms over a broad range of plasmid size. Static light scattering data were obtained using supercoiled, open‐circular, and linear isoforms of 5.76, 9.80, and 16.8 kbp plasmids. The results from this study extend the range of R_G values available in the literature to plasmid sizes typically used for gene therapy and DNA vaccines. The experimental data were compared with available theoretical expressions based on the worm‐like chain model, with the best‐fit value of the apparent persistence length for both the linear and open‐circular isoforms being statistically identical at 46 nm. A new expression was developed for the radius of gyration of the supercoiled plasmid based on a model for linear DNA using an effective contour length that is equal to a fraction of the total contour length. These results should facilitate the development of micro/nano‐fluidic devices for DNA manipulation and size‐based separation processes for plasmid DNA purification. Biotechnol. Bioeng. 2010;107: 134–142. © 2010 Wiley Periodicals, Inc.     

Role of hydrophobicity in protein structure is overestimated

Some microcalorimetrically measured and theoretically calculated thermodynamic interaction coefficients of amino acid compounds in aqueous and peptidic systems have been collected, and are reported here. They indicate that although hydrophobicity contributes to the stability of the native structure of proteins, its influence on their exact configuration is limited.     

Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima

Studies of the structural basis of protein thermostability have produced a confusing picture. Small sets of proteins have been analyzed from a variety of thermophilic species, suggesting different structural features as responsible for protein thermostability. Taking advantage of the recent advances in structural genomics, we have compiled a relatively large protein structure dataset, which was constructed very carefully and selectively; that is, the dataset contains only experimentally determined structures of proteins from one specific organism, the hyperthermophilic bacterium Thermotoga maritima, and those of close homologs from mesophilic bacteria. In contrast to the conclusions of previous studies, our analyses show that oligomerization order, hydrogen bonds, and secondary structure play minor roles in adaptation to hyperthermophily in bacteria. On the other hand, the data exhibit very significant increases in the density of salt-bridges and in compactness for proteins from T.maritima. The latter effect can be measured by contact order or solvent accessibility, and network analysis shows a specific increase in highly connected residues in this thermophile. These features account for changes in 96% of the protein pairs studied. Our results provide a clear picture of protein thermostability in one species, and a framework for future studies of thermal adaptation.     

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.     

A novel correlation for protein diffusion coefficients based on molecular weight and radius of gyration

A new correlation is proposed for the prediction of protein diffusion coefficients in free solution. Molecular weight and radius of gyration of proteins are employed as correlation parameters in this method. Both parameters can be easily found in the literature. The correlation works well for diverse proteins with different shapes and extensive molecular weight. Furthermore, this method does not require a preassumption regarding the protein shape while it offers a rapid and convenient calculation with a high accuracy. Also, the proposed correlation can elucidate the estimation deviation of previous correlation methods in the literature.     

Periodicity in DNA primary structure is defined by secondary structure of the coded protein.

A 10.5-base periodicity found earlier is inherent in both eu- and prokaryotic coding nucleotide sequences. In the case of noncoding eukaryotic sequences no periodicity is found, so the 10.5-base oscillation seemingly does not correlate with the nucleosomal organization of DNA. It is shown that the DNA fragments, coding the alpha-helical protein segments, manifest the pronounced 10.5-base periodicity, while those regions of DNA which code the beta-structure have a 6-base oscillation. The repeating pattern of nucleotide sequences can be used for comparison of the DNA segments with low degree of homology.     

Quantitative prediction of protein secondary structure —Where is the lacuna?

We make a brief review of the available methods to predict, in particular, secondary structures of proteins quantitatively, not only from theoretical considerations but also from various spectroscopic techniques, notably, CD,IR, Raman, and NMR. Since the quantitative data obtained on secondary structures, such as helices, sheets, turns, random coils, and unordered structures, are always compared to the available X-ray data as a validity of the method, we show from published results and from simple calculations on nearly twenty proteins that there can be substantial differences in the final results, depending on the criteria used for calculations. We point out the need for i) taking into account the solvent-induced distortion of the secondary structures, ii) carrying out complementary experiments under different physico-chemical conditions to be reasonably confident about peak assignments, and iii) calculating vibrational modes, preferably in the range of frequencies that are easily accessible to the spectroscopists. We also urge reduction of the current overemphasis of numerical analysis of spectral profiles without proper physico-chemical rationale.     

The liganding of glycolipid transfer protein is controlled by glycolipid acyl structure

Glycosphingolipids (GSLs) play major roles in cellular growth and development. Mammalian glycolipid transfer proteins (GLTPs) are potential regulators of cell processes mediated by GSLs and display a unique architecture among lipid binding/transfer proteins. The GLTP fold represents a novel membrane targeting/interaction domain among peripheral proteins. Here we report crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure and occupancy of the hydrophobic tunnel dictate partitioning between sphingosine-in and newly-observed sphingosine-out ligand-binding modes. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to and/or from membranes, as well as during GSL presentation and/or transfer to other proteins.     

Secondary structure of filamentous bacteriophage coat protein is preserved in lipid environments

1H nuclear magnetic resonance experiments have shown that the amide hydrogens of residues 30 to 40 of bacteriophage Pf1 coat protein in micelles undergo very slow exchange with solvent deuterons. The amide 1H resonances from these residues were used to monitor the structural stability of the membrane-spanning helix of the coat protein during the transition of the coat protein from its structural form, in the virus particle, to the membrane-bound form, in micelles. The helix was found to remain folded on the 10(-3) second time-scale of the experiment, which indicates that no major disruption or rearrangement of the central part of the protein structure occurs during the process of coat protein solubilization by detergent. The results also suggest that a helical peptide can associate with lipids without reorganization of its secondary structure. However, a general model for the insertion of proteins into membranes cannot be established from these results, because the mechanism of the detergent solubilization process may differ somewhat from that of the membrane insertion process.     

Unusual mRNA pseudoknot structure is recognized by a protein translational repressor

Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.     

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left arm of the second chromosome at position 28D2-4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2A alpha or PP2A beta, indicating that PP2A may be the most conserved of all known enzymes.     

Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

The structure of protein evolution and the evolution of protein structure

The observed distribution of protein structures can give us important clues about the underlying evolutionary process, imposing important constraints on possible models. The availability of results from an increasing number of genome projects has made the development of these models an active area of research. Models explaining the observed distribution of structures have focused on the inherent functional capabilities and structural properties of different folds and on the evolutionary dynamics. Increasingly, these elements are being combined.     

Radius construction and structure in the orb-web of Zilla diodia (Araneidae)

In orb-webs, the tension of the sticky spiral produces a centripetal force on the radii, resulting in an increase in tension along each radius from the centre of the web to the periphery. Zilla diodia (Walckenaer, 1802) atypical of araneids, was found to adapt the structure of its radii to this tension gradient by building radii that are double stranded at the periphery of the web and single stranded near the centre. Furthermore, the proportion of each radius that is doubled was found to be larger in the upper part of the web - where the overall tensions in the radii are known to be higher than in the lower part of the web. suggesting that the spider adjusts the proportion of each radius that is doubled to the overall tension in the radius.     

The apparent Km is a misleading kinetic indicator.

When information concerning whether or not a ligand interacts with the same enzyme species as do the substrates, the variation of the Michaelis constant Km (for each substrate) with ligand concentration is sometimes used as a diagnostic. It is shown that the Michaelis constant is of no particular value in this respect and may be misleading. Thus, depending on the mechanism, Km may vary with ligand concentration even though the ligand interacts with species far removed in the mechanism from the substrate-binding steps, and it may stay constant in cases where the ligand competes directly for the free enzyme. In contrast, the slope of a double-reciprocal plot of the kinetic data (= Km/Vmax.) (or, equivalently, the ordinate intercept of a Hanes plot A/v versus A, where A is the substrate concentration) independently of the particular mechanism involved uniquely signifies whether or not such interaction occurs. The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope, in accordance with the long-established rules of Cleland [Biochim. Biophys. Acta (1963) 67, 188-196], for which the present analysis provides the formal framework.