Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g? conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oγ atom of Ser and Thr and the backbone carbonyl oxygen at position i ? 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the β₂-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.     

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Fo?rster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 ?) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

What is the role of thermodynamics on protein stability?

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article.     

Influence of a few coat protein subunits on the base-paired structure of 3′-terminal fragments of RNA 4 of alfalfa mosaic virus

In contrast to expectation (Srinivasan, S. and Jaspars, E.M.J. (1978) Biochim. Biophys. Acta 520, 237–241) differentiated thermal melting profiles and fluorescence measurements show that the coat protein of alfalfa mosaic virus has a negligible effect on the base-paired structure of isolated 3′-terminal fragments (length about 90 nucleotides) of the coat protein messenger RNA (RNA 4) of this virus.     

Influence of organization of native protein structure on its folding: Modeling of the folding of α-helical proteins

An important question that is addressed here is whether the modeling of protein folding can catch the difference between the folding of proteins with similar structures but with different folding mechanisms. In this work, the modeling of folding of four α-helical proteins from the homeodomain family, which are similar in size, was done using the Monte Carlo and dynamic programming methods. A frequently observed order of folding of α-helices for each protein was determined using the Monte Carlo method. A correlation between the experimental folding rate and the number of Monte Carlo steps was also demonstrated. Amino acid residues that are important for the folding were determined using the dynamic programming method. The defined regions correlate with the order of folding of secondary-structure elements in the proteins both in experiments and in modeling.     

Studies on the rules of β-strand alignment in a protein β-sheet structure

To further disclose the underlying mechanisms of protein β-sheet formation, studies were made on the rules of β-strands alignment forming β-sheet structure using statistical and machine learning approaches. Firstly, statistical analysis was performed on the sum of β-strands between each β-strand pairs in protein sequences. The results showed a propensity of near-neighbor pairing (or called “first come first pair”) in the β-strand pairs. Secondly, based on the same dataset, the pairwise cross-combinations of real β-strand pairs and four pseudo-β-strand contained pairs were classified by support vector machine (SVM). A novel feature extracting approach was designed for classification using the average amino acid pairing encoding matrix (APEM). Analytical results of the classification indicated that a segment of β-strand had the ability to distinguish β-strands from segments of α-helix and coil. However, the result also showed that a β-strand was not strongly conserved to choose its real partner from all the alternative β-strand partners, which was corresponding with the ordination results of the statistical analysis each other. Thus, the rules of “first come first pair” propensity and the non-conservative ability to choose real partner, were possible important factors affecting the β-strands alignment forming β-sheet structures.     

Effect of glycerol–water binary mixtures on the structure and dynamics of protein solutions

We have performed 20?ns of fully atomistic molecular dynamics simulations of Hen Egg-White Lysozyme in 0, 10, 20, 30, and 100% by weight of glycerol in water to better understand the microscopic physics behind the bioprotection offered by glycerol to naturally occuring biological systems. The solvent exposure of protein surface residues changes when glycerol is introduced. The dynamic behavior of the protein, as quantified by the incoherent intermediate scattering function, shows a nonmonotonic dependence on glycerol content. The fluctuations of the protein residues with respect to each other were found to be similar in all water-containing solvents, but different from the pure glycerol case. The increase in the number of protein–glycerol hydrogen bonds in glycerol–water binary mixtures explains the slowing down of protein dynamics as the glycerol content increases. We also explored the dynamic behavior of the hydration layer. We show that the short length scale dynamics of this layer are insensitive to glycerol concentration. However, the long length scale behavior shows a significant dependence on glycerol content. We also provide insights into the behavior of bound and mobile water molecules.     

The structure of the elicitor Cerato-platanin (CP), the first member of the CP fungal protein family, reveals a double ψβ-barrel fold and carbohydrate binding

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψβ-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the β-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψβ-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.     

What is the minimum number of letters required to fold a protein?

Experimental studies have shown that the full sequence complexity of naturally occurring proteins is not required to generate rapidly folding and functional proteins, i.e. proteins can be designed with fewer than 20 letters. This raises the question of what is the minimum number of amino acid types required to encode complex protein folds? Here, we investigate this issue from three aspects. First, we study the minimum sequence complexity that can reserve the necessary structural information for detection of distantly related homologues. Second, we compare the ability of designing foldable model sequences over a wide range of reduced amino acid alphabets, which find the minimum number of letters that have the similar design ability as 20. Finally, we survey the lower bound of alphabet size of globular proteins in a non-redundant protein database. These different approaches give a remarkably consistent view, that the minimum number of letters required to fold a protein is around ten.     

Influence of C-H...O interactions on the structural stability of β-lactamases

β-Lactamases produced by pathogenic bacteria cleave β-lactam antibiotics and render them ineffective. Understanding the principles that govern the structural stability of β-lactamases requires elucidation of the nature of the interactions that are involved in stabilization. In the present study, we systematically analyze the influence of CH...O interactions on determining the specificity and stability of β-lactamases in relation to environmental preferences. It is interesting to note that all the residues located in the active site of β-lactamases are involved in CH...O interactions. A significant percentage of CH...O interactions have a higher conservation score and short-range interactions are the predominant type of interactions in β-lactamases. These results will be useful in understanding the stability patterns of β-lactamases.     

The effect of a Pro²⁸Thr point mutation on the local structure and stability of human galactokinase enzyme-a theoretical study

Galactokinase is responsible for the phosphorylation of α-d-galactose, which is an important step in the metabolism of the latter. Malfunctioning of galactokinase due to a single point mutation causes cataracts and, in serious cases, blindness. This paper reports a study of the Pro²⁸Thr point mutation using a variety of theories including molecular dynamics (MD), MM-PBSA/GBSA calculations and AIM analysis. Altered H-bonding networks were detected based on geometric and electron density criteria that resulted in local unfolding of the β-sheet secondary structure. Another consequence was the decrease in stability (5–7 kcal mol⁻¹) around this region, as confirmed by ΔG_bind calculations for the extracted part of the whole system. Local unfolding was verified by several other MD simulations performed with different duration, initial velocities and force field. Based on the results, we propose a possible mechanism for the unfolding caused by the Pro²⁸Thr point mutation.     

Influence of Flexibility and Variability of Working Hours on Health and Well‐Being

Flexible working hours can have several meanings and can be arranged in a number of ways to suit the worker and/or employer. Two aspects of “flexible” arrangement of working hours were considered: one more subjected to company control and decision (variability) and one more connected to individual discretion and autonomy (flexibility). The aim of the study was to analyze these two dimensions in relation to health and well‐being, taking into consideration the interaction with some relevant background variables related to demographics plus working and social conditions. The dataset of the Third European Survey on working conditions, conducted in 2000 and involving 21,505 workers, was used. Nineteen health disorders and four psycho‐social conditions were tested by means of multiple logistic regression analysis, in which mutually adjusted odds ratios were calculated for age, gender, marital status, number of children, occupation, mode of employment, shift work, night work, time pressure, mental and physical workload, job satisfaction, and participation in work organization. The flexibility and variability of working hours appeared inversely related to health and psycho‐social well‐being: the most favorable effects were associated with higher flexibility and lower variability. The analysis of the interactions with the twelve intervening variables showed that physical work, age, and flexibility are the three most important factors affecting health and well‐being. Flexibility resulted as the most important factor to influence work satisfaction; the second to affect family and social commitment and the ability to do the same job when 60 years old, as well as trauma, overall fatigue, irritability, and headache; and the third to influence heart disease, stomachache, anxiety, injury, and the feeling that health being at risk because of work. Variability was the third most important factor influencing family and social commitments. Moreover, shift and night work confirmed to have a significant influence on sleep, digestive and cardiovascular troubles, as well and health and safety at work. Time pressure also showed a relevant influence, both on individual stress and social life. Therefore, suitable arrangements of flexible working time, aimed at supporting workers' coping strategies, appear to have a clear beneficial effect on worker health and well‐being, with positive consequences also at the company and social level, as evidenced by the higher “feeling to be able to work until 60 years of age”.     

Improving the stability of thiol–maleimide bioconjugates via the formation of a thiazine structure

Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol–maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement. Our preliminary work indicated that the thiazine linker has favorable stability. Here, we report the evaluation of a thiazine linker as an alternative to the widely used succinimidyl thioether linker for thiol–maleimide bioconjugation. The stability of the thiazine conjugate in comparison to the thioether conjugate was assessed across a broad pH range. Additionally, the propensity for retro-Michael reaction and cross-reactivity with other thiols was evaluated by treating conjugates in the presence of glutathione. The studies indicated that the thiazine linker degrades markedly slower than the thioether conjugate. In addition, the thiazine linker is over 20 times less susceptible to glutathione adduct formation. The NMR study of the thiazine structure confirmed that the formation of the thiazine linker is a stereoselective process that yields a single diastereomer. In summary, we propose the use of the thiazine linker obtained by conjugation of maleimide-containing reagents with peptides or proteins presenting an N-terminal cysteine as a novel approach for bioconjugation. The advantages of this approach are the formation of a linker with a well-defined stereochemical configuration, increased stability at physiological pH, and a strongly reduced propensity for thiol exchange.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

Influence of 4′-6-diamidino-2-phenylindole on the secondary structure and template activities of DNA and polydeoxynucleotides

Summary The interaction between 4-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI-DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380–392 nm for DAPI-DNA complexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex.Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.     

Free energy calculations on the stability of the 14-3-3ζ protein

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.     

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein,an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.     

Remarks on the internal structure of the shells of Ammonoidea,Gastropoda and Foraminifera — with a short introduction on biostratigraphy and paleo-ecology

The modern principles of marine biostratigraphy ask for more exact fossil determinations. In view of the increased activity in nearly all countries concerning this modern trend in geology more uniformity in the methods used for the determination of fossils and especially of microfossils, is needed. In this way a better understanding of the stratigraphical range of important species and their natural relations to other species in all parts of the world would be obtained. For this purpose the study of the internal structures is also important: siphuncular structures of the ammonite shell—heterostrophical oldest volutions of gastropods—canal systems of foraminifers. A few examples are given and figured. A few remarks on biostratigraphy, paleo-ecology, facies and guide fossils serve as an introduction.