Stability Of Protein Secondary Structure Research Articles

Effects of protein oxidation on the rheological behaviour of different wheat flour

AbstractWheat flour proteins are subject to oxidation reactions during production, processing and storage. The quality of protein and the rheological properties of wheat are crucial for the flour industry. However, the impact and mechanism of protein oxidised on wheat flour quality remain unclear. In this study, ozone was used to oxidise wheat grains, the secondary structure of protein in flour and the rheological properties of dough were analysed by FTIR and Mixolab. The proportion of α‐helix and β‐folding of protein were decreased significantly, as were the development time (DDT), stability time (DST) and protein weakening (C2) value of dough. Meanwhile, starch gelatinisation (C3), amylase activity (C4) and retrogradation (C5) were increased significantly, along with the elastic modulus (G′) and viscous modulus (G″). Microstructure analysis indicated that protein oxidation destroyed the gluten network structure in the dough. In addition, the L* value of dough was decreased and a* and b* values were increased significantly. The results showed that the oxidation of protein reduced the stability of protein secondary structure, weakened the structure and stability of the gluten network in dough, and changed the viscoelasticity and colour of dough. Overall, these findings provide a better understanding of rheological behaviour in wheat flour.

Plasma membrane aquaporins mediates vesicle stability in broccoli.

The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.

Thermostability of photosystem I trimers and monomers from the cyanobacterium Thermosynechococcus elongatus

The performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermostability of photosystem I (PSI) complexes Terasaki et al. (2007), Iwuchukwu et al. (2010), Kothe et al. (2013) . To assess the thermostability of PSI complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus heating induced perturbations on the level of secondary structure of the proteins were studied. Changes were monitored by Fourier transform infrared (FT-IR) spectra in the mid-IR region upon slow heating (1°C per minute) of samples in D2O phosphate buffer (pD 7.4) from 20°C to 100°C. These spectra showed distinct changes in the Amide I region of PSI complexes as a function of the rising temperature. Absorbance at the Amide I maximum of PSI monomers (centered around 1653cm−1), gradually dropped in two temperature intervals, i.e. 60–75 and 80–90°C. In contrast, absorbance at the Amide I maximum of PSI trimers (around 1656cm−1) dropped only in one temperature interval 80–95°C. The thermal profile of the spectral shift of α-helices bands in the region 1656–1642cm−1 confirms the same two temperature intervals for PSI monomers and only one interval for trimers. Apparently, the observed absorbance changes at the Amide I maximum during heating of PSI monomers and trimers are caused by deformation and unfolding of α-helices. The absence of absorbance changes in the interval of 20–65°C in PSI trimers is probably caused by a greater stability of protein secondary structure as compared to that in monomers. Upon heating above 80°C a large part of α-helices both in trimers and monomers converts to unordered and aggregated structures. Spectral changes of PSI trimers and monomers heated up to 100°C are irreversible due to protein denaturation and non-specific aggregation of complexes leading to new absorption bands at 1618–1620cm−1. We propose that monomers shield the denaturation sensitive sides at the monomer/monomer interface within a trimer, making the oligomeric structure more stable against thermal stress.

Precise structural analysis of α-helical polypeptide by quantum-chemical calculation related to reciprocal side-chain combination of two L-phenylalanine residues

To clarify the positive role of side-chain conformation in the stability of protein secondary structure (main-chain conformation), we successfully calculated the optimization structure of a series of well-defined α-helical octadecapeptides composed of two L-phenylalanine (Phe) and 16 L-alanine (Ala) residues, based on the molecular orbital calculation with density functional theory (DFT/B3LYP/6-31G(d)). From the total energy calculation and the precise secondary structural analysis, we found that the conformational stability of the α-helix is closely related to the reciprocal side-chain combinations (such as positional relation and side-chain conformation) of two Phe residues in this system. Furthermore, we demonstrated that the 1H, 13C, 15N and 17O isotropic chemical shifts of each Phe residue depend on the respective side-chain conformations of the Phe residue.

Precise side-chain conformation analysis of l-phenylalanine in α-helical polypeptide by quantum-chemical calculation and 13C CP-MAS NMR measurement

To clarify the positive role of side-chain conformation in the stability of protein secondary structure (main-chain conformation), we successfully calculated the optimization structure of a well-defined α-helical octadecapeptide composed of l-alanine (Ala) and l-phenylalanine (Phe) residues, H–(Ala) 8–Phe–(Ala) 9–OH, based on the molecular orbital calculation with density functional theory (DFT/B3LYP/6-31G(d)). From the total energy and the precise secondary structural parameters such as main-chain dihedral angles and hydrogen-bond parameters of the optimized structure, we confirmed that the conformational stability of an α-helix is affected dominantly by the side-chain conformation ( χ 1) of the Phe residue in this system: model A ( T form: around 180° of χ 1) is most stable in α-helix and model B ( G + form: around −60° of χ 1) is next stable, but model C ( G − form: around 60° of χ 1) is less stable. In addition, we demonstrate that the stable conformation of poly( l-phenylalanine) is an α-helix with the side-chain T form, by comparison of the carbonyl 13C chemical shift measured by 13C CP-MAS NMR and the calculated one.

Salt-Specific Stability and Denaturation of a Short Salt-Bridge-Forming α-Helix

The structure of a single alanine-based Ace-AEAAAKEAAAKA-Nme peptide in explicit aqueous electrolyte solutions (NaCl, KCl, NaI, and KF) at large salt concentrations (3-4 M) is investigated using approximately 1 mus molecular dynamics (MD) computer simulations. The peptide displays 71% alpha-helical structure without salt and destabilizes with the addition of NaCl in agreement with experiments of a somewhat longer version. It is mainly stabilized by direct and indirect (" i + 4")EK salt bridges between the Lys and Glu side chains and a concomitant backbone shielding mechanism. NaI is found to be a stronger denaturant than NaCl, while the potassium salts hardly show influence. Investigation of the molecular structures reveals that consistent with recent experiments Na (+) has a much stronger affinity to side chain carboxylates and backbone carbonyls than K (+), thereby weakening salt bridges and secondary structure hydrogen bonds. At the same time, the large I (-) has a considerable affinity to the nonpolar alanine in line with recent observations of a large propensity of I (-) to adsorb to simple hydrophobes, and thereby "assists" Na (+) in its destabilizing action. In the denatured states of the peptide, novel long-lived (10-20 ns) "loop" configurations are observed in which single Na (+) ions and water molecules are hydrogen-bonded to multiple backbone carbonyls. In an attempt to analyze the denaturation behavior within the preferential interaction formalism, we find indeed that for the strongest denaturant, NaI, the protein is least hydrated. Additionally, a possible indication for protein denaturation might be a preferential solvation of the peptide backbone by the destabilizing cosolute (sodium). The mechanisms found in this work may be of general importance to understand salt effects on protein secondary structure stability.

Rapid and accurate processing method for amide proton exchange rate measurement in proteins

Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing.

Resveratrol Binding to Human Serum Albumin

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of KRes = 2.56× 105 M−1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.

Retinol and retinoic acid bind human serum albumin: Stability and structural features

Vitamin A components, retinol and retinoic acid, are fat-soluble micronutrients and critical for many biological processes, including vision, reproduction, growth, and regulation of cell proliferation and differentiation. The cellular uptake of Vitamin A is through specific interaction of a plasma membrane receptor with serum retinol-binding protein. Human serum albumin (HSA), as a transport protein, is the major target of several micronutrients in vivo. The aim of present study was to examine the interaction of retinol and retinoic acid with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various retinoid contents. FTIR, UV–vis, CD and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant and the effects of complexation on protein secondary structure. Structural analysis showed that retinol and retinoic acid bind non-specifically (H-bonding) via protein polar groups with binding constants of K ret = 1.32 (±0.30) × 10 5 M −1 and K retac = 3.33 (±0.35) × 10 5 M −1. The protein secondary structure showed no alterations at low retinoid concentrations (0.125 mM), whereas at high retinoid content (1 mM), an increase of α-helix from 55% (free HSA) to 60% and a decrease of β-sheet from 22% (free HSA) to 18% occurred in the retinoid–HSA complexes. The results point to a partial stabilization of protein secondary structure at high retinoid content.

Tyrosine 387 and Arginine 404 Are Critical in the Hydrolytic Mechanism of Escherichia coli Aminopeptidase P

Analysis of the pH-rate profile for catalysis of bradykinin cleavage by aminopeptidase P (AMPP), a manganese-containing hydrolase from Escherichia coli, was carried out to show that optimal catalytic function is obtained at neutral pH. On the basis of information derived from the crystal structure, peptidase sequence alignments, and the hydrolysis of organophosphate triesters, active site residues Arg153, Arg370, Trp88, Tyr387, and Arg404 were identified as potential catalytic residues. Site-directed mutagenesis was used to substitute these residues with Leu, Ala, Trp, Lys, or Phe. The kcat values for the Arg153, Arg370, and Trp88 mutants were nearly the same as that for the wild-type enzyme. The kcat values of the R404K, R404A, and Y387A mutants were lower by factors of 285, 400, and 16, respectively. Inductively coupled plasma mass spectrometry and circular dichroism spectroscopy showed that Arg404 is not required for metal chelation or stabilization of protein secondary structure. The hydrogen bond network observed between the side chains of conserved residues Asp260, Arg404, and Tyr387 indicated that Arg404 participates in proton relay. This was further evidenced by the return of activity in the R404A mutant by the addition of guanidine. Also, reduced catalytic efficiency in the R404K mutant, which conserves the positive charge at the bridge site, shows that only the arginine group of Arg404 (not the ammonium group of Lys404) can participate in the hydrogen bond network. The hydrogen bond interaction between the Arg404 and the Tyr387 ring hydroxyl group is suggested by the reduced catalytic efficiency of the Y387F mutant.

Liquid NMA: A surprisingly realistic model for hydrogen bonding motifs in proteins

The structure of liquid N-methylacetamide is examined using a combination of classical and ab initio molecular dynamics. Polymeric hydrogen bonded (N–H⋯O C) chain motifs having complex topologies emerge as the dominant structural features. Interchain associations, reminiscent of β-sheets in proteins, are also evident and may be stabilized by improper (methyl-donated) hydrogen bonds. Both ab initio and classical simulations lead to a consistent picture of local structure, including the presence of close contacts between oxygen and methyl hydrogen atoms. Liquid NMA, therefore, exhibits a surprisingly complex structure, and possesses two clear bonding motifs relevant to the stability of protein secondary structure.

Long-Term Stability of Protein Secondary Structure in Dry Seeds

Abstract Changes in protein secondary structure during seed aging were studied by in situ Fourier transform infrared microspectroscopy. Seeds of onion, white cabbage and radish, harvested in 1969, were stored at 15–20°C and 30% relative humidity. Fresh control seeds were harvested in 1994 and stored under the same conditions. In 1995, the germination capacity of seeds was >90% for the 1994 harvest and zero for the 1969 harvest. Inspection of the amide-I bands in Fourier self-deconvolved infrared spectra of thin slices of embryo axes of the various seeds did not reveal major changes in relative peak height and band position of the different protein secondary structures with aging. No protein aggregation and denaturation were found after long-term storage. Heating of dry viable radish seeds up to 145°C did not cause appreciable protein denaturation. In contrast, boiling hydrated radish seeds for 5 min led to decoiling of the α-helical structure and the appearance of a band at 1627 cm−1. This band is characteristic of intermolecular extended β-sheet structures. We conclude that, despite the loss of viability and the long postmortem storage period, secondary structure of proteins in desiccation tolerant dry seed is very stable and conserved during several decades of open storage.

Thermal stability of protein secondary structure in Langmuir-Blodgett films

The temperature dependence of the secondary structure of photosynthetic reaction centres from Rhodobacter sphaeroides in solution and in Langmuir-Blodgett film was studied by circular dichroism. It was shown that the secondary structure of the protein was not affected in Langmuir-Blodgett films by heating up to 200°C, while in solution it was completely lost at 55°C. Molecular order rather than decreased hydration degree was held responsible.

Fourier transform infrared spectroscopy for the characterization of a model peptide–DNA interaction

To better understand the structural basis of protein-DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29-residue synthetic peptide binds nonspecifically to heterogeneous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100-200 microM produces 50% saturation of heterogeneous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second-derivative spectra resolve the amide I band of native peptide into components located at 1627 (beta-strand), 1658 (alpha-helix), and 1681 (turn or beta-strand) cm-1, with a distinct shoulder at 1647 cm-1 (disordered structure). Assignment of the 1681 cm-1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and beta-strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm-1 (turn) is no longer seen; a new band appears at 1675 cm-1; the 1627 cm-1 band (beta-strand) is considerably reduced in intensity; the position of the alpha-helical (1658 cm-1) component remains unchanged; the shoulder at 1647 cm-1 (disorder) disappears. The new vibration at 1675 cm-1 is characteristic of beta-strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm-1 (bound); the relative intensities of vAS and the PO2- symmetric stretch (vS) are altered upon peptide binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II

As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55–77 and 56–77 appeared first, followed by peptides of residues 4–23, 29–39, 40–44 and 45–54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl- sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.

Ion pairs in alpha helices.

A survey of 47 globular proteins was made to determine the probability of occurrence of ion pairs separated by 1,2,3,... and 8 residues in the alpha helices. As a control, the probability of occurrence of like charged pairs was also determined. The survey showed that ion pairs of the type i,i +/- 3 and i, i +/- 4 are the most predominant. Such a preference was not observed for like charged pairs. The observed frequency of ion pairs is significantly greater than their expected frequency. The normalized frequencies of occurrence of the ion pairs were also found to increase generally with the helix length. These results indicate that the ion pairs may contribute to the stability of solvent-exposed alpha helices. Since the stabilization of protein secondary structure enhances the stability of protein tertiary structure, these results may throw light on the mechanism of protein folding.

Stabilization of the long central helix of troponin C by intrahelical salt bridges between charged amino acid side chains.

The unusual dumbbell shape of troponin C is due to the presence of a long alpha-helix of nine turns that connects the amino- and carboxyl-terminal calcium-binding domains. The center of the long helix appears to be stabilized by several salt bridges. The long helix is also bent about 16 degrees at glycine-92. Calmodulin, which lacks the central glycine, also is predicted to be stabilized by salt bridges in the central helix. The presence of a proline residue in the center of the long helix of ascidian troponin C and the myosin regulatory light chains suggests that a sharper bend may occur in these molecules. The conservation of the bend and salt bridges in the related calcium-binding proteins suggests they may have an important biological function. The structure of troponin C suggests that intrahelix salt bridges between neighboring charged residues may be involved in the stabilization of protein secondary structure. The preponderance of potential salt bridges in other muscle proteins as well may be related to their elongated structures and their participation in the contractile process.

Genetic code redundancy and the evolutionary stability of protein secondary structure

The genetic code has an inherent bias towards some amino acids because of the variable number of synonymous codons per amino acid. The extent to which these biases are expressed in protein secondary structure is described through the analysis of the overall amino acid compositions of the alpha-helix, beta-sheet, beta-turn and random coil segments elucidated by X-ray crystallography. Given the concept of neutral mutation in proteins, the allocation of synonyms in the genetic code appears to protect secondary structures from amino acid changes and discourages the appearance of chemically complex residues. The level of protection is similar for each structural form, despite their clear preferences for certain amino acids. The organization of the code is therefore relevant to the preservation of conformation seen in the evolution of many protein families.