Detection of Structural Changes of Enzymes in Nonaqueous Media by Fluorescence and CD Spectroscopy

SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

Download Full-text

Chasing Particularities of Guanine- and Cytosine-Rich DNA Strands

Molecules ◽

10.3390/molecules25030434 ◽

2020 ◽

Vol 25 (3) ◽

pp. 434 ◽

Cited By ~ 4

Author(s):

Marko Trajkovski ◽

Janez Plavec

Keyword(s):

Structural Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Dna Recombination ◽

Cd Spectroscopy ◽

G Quadruplex ◽

Recombination Processes ◽

Dna Strands ◽

Native Polyacrylamide Gel Electrophoresis ◽

Myc Gene

By substitution of natural nucleotides by their abasic analogs (i.e., 1′,2′-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson–Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

Download Full-text

A review of the interaction between anthocyanins and proteins

Food Science and Technology International ◽

10.1177/1082013220962613 ◽

2020 ◽

pp. 108201322096261

Author(s):

Jia Li ◽

Bixiang Wang ◽

Yang He ◽

Liankui Wen ◽

Hailong Nan ◽

...

Keyword(s):

Activation Energy ◽

Structural Changes ◽

Hydrophobic Interactions ◽

Cd Spectroscopy ◽

Hydrogen Bonding Interactions ◽

The Stability ◽

Α Helix ◽

Proteins Interactions ◽

Β Sheet ◽

Different Sources

Anthocyanins have good physiological functions, but they are unstable. The interaction between anthocyanins and proteins can improve the stability, nutritional and functional properties of the complex. This paper reviews the structural changes of complex of anthocyanins interacting with proteins from different sources. By circular dichroism (CD) spectroscopy, it was found that the contents of α-helix (from 15.90%−42.40% to 17.60%−52.80%) or β-sheet (from 29.00%−50.00% to 29.40%−57.00%) of the anthocyanins–proteins complex increased. Fourier transform infrared spectroscopy showed that the regions of amide I (from 1627.87−1641.41 cm−1 to 1643.34−1651.02 cm−1) and amide II (from 1537.00−1540.25 cm−1 to 1539.00−1543.75 cm−1) of anthocyanins–proteins complex were shifted. Fluorescence spectroscopy showed that the fluorescence intensity of the complex decreased from 150−5100 to 40−3900 a.u. The thermodynamic analysis showed that there were hydrophobic interactions, electrostatic and hydrogen bonding interactions between anthocyanins and proteins. The kinetic analysis showed that the half-life and activation energy of the complex increased. The stability, antioxidant, digestion, absorption, and emulsification of the complex were improved. This provides a reference for the study and application of anthocyanins and proteins interactions.

Download Full-text

Effect of cosolvents (polyols) on structural and foaming properties of soy protein isolate

Czech Journal of Food Sciences ◽

10.17221/35/2016-cjfs ◽

2017 ◽

Vol 35 (No. 1) ◽

pp. 57-66 ◽

Cited By ~ 3

Author(s):

Pan Mingzhe ◽

Meng Xianjun ◽

Jiang Lianzhou ◽

Yu Dianyu ◽

Liu Tianyi

Keyword(s):

Soy Protein ◽

Structural Changes ◽

Foam Stability ◽

Soy Protein Isolate ◽

Tryptophan Fluorescence ◽

Surface Hydrophobicity ◽

Cd Spectroscopy ◽

Protein Isolate ◽

Foaming Properties ◽

Intrinsic Tryptophan Fluorescence

Effect of polyols (mannitol, sorbitol, and xylitol) at three concentrations (5, 10, and 15% w/w) on the structure of soy protein isolates (SPI) was investigated. Changes in foaming properties of SPI were then examined with the addition of polyols at different concentrations. The interactions between SPI and polyols resulted in a substantial decrease in protein surface hydrophobicity and intrinsic tryptophan fluorescence intensity, along with the covering of tyrosine. Furthermore, circular dichroism (CD) spectroscopy of SPI suggested that a more ordered and compact conformation was induced by polyols. Consequently, these structural changes led to lower foamability of SPI. An increase in the viscosity of SPI suspension seemed to be advantageous for improving the foam stability of SPI.

Download Full-text

Contribution of Chemical Modifications and Conformational Epitopes to IgE Binding by Ara h 3

Foods ◽

10.3390/foods7110189 ◽

2018 ◽

Vol 7 (11) ◽

pp. 189 ◽

Cited By ~ 3

Author(s):

Scott Dyer ◽

Jacqueline Nesbit ◽

Beatriz Cabanillas ◽

Hsiaopo Cheng ◽

Barry Hurlburt ◽

...

Keyword(s):

Heat Treatment ◽

Secondary Structure ◽

Disulfide Bonds ◽

Structural Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cd Spectroscopy ◽

Chemical Modifications ◽

Ige Binding ◽

Ara H 3

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

Download Full-text

Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding

Biochemical Journal ◽

10.1042/bj3350319 ◽

1998 ◽

Vol 335 (2) ◽

pp. 319-327 ◽

Cited By ~ 16

Author(s):

Peter MACHEROUX ◽

Ernst SCHÖNBRUNN ◽

Dmitri I. SVERGUN ◽

Vladimir V. VOLKOV ◽

Michel H. J. KOCH ◽

...

Keyword(s):

Ternary Complex ◽

Structural Changes ◽

Tertiary Structure ◽

Tryptophan Fluorescence ◽

Cd Spectroscopy ◽

Scattering Data ◽

Discrete State ◽

Chorismate Synthase ◽

X Ray Scattering ◽

Ft Ir

Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover. This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences. It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein. Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state. The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little. Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein. Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an α–β-barrel using discrete state-space modelling.

Download Full-text

Structural changes of protein antigens due to adsorption onto and release from aluminium hydroxide using FTIR–ATR

Spectroscopy An International Journal ◽

10.1155/2007/354051 ◽

2007 ◽

Vol 21 (4) ◽

pp. 211-226 ◽

Cited By ~ 7

Author(s):

Yiwu Zheng ◽

Xuxin Lai ◽

Henrik Ipsen ◽

Jørgen Nedergaard Larsen ◽

Henning Løwenstein ◽

...

Keyword(s):

Immune Response ◽

Aluminium Hydroxide ◽

Structural Changes ◽

Structural Integrity ◽

Structural Information ◽

Cd Spectroscopy ◽

Attenuated Total Reflection ◽

Protein Antigens ◽

Recovery Curves ◽

Atr Spectroscopy

Structural integrity of antigens upon adsorption and release is not only important for investigating vaccine immunogenicity, but also for the epitope specificity of the resulting immune response and hence therapeutic efficacy. Moreover, the structural information is also important for understanding the mechanism of how adjuvants can enhance the immune response. However, little is known about an antigen's structure when it is adsorbed on and subsequently released from aluminium adjuvants. In this study, the structures of two protein antigens, bovine serum albumin and β-lactoglobulin, were investigated using Fourier transform infrared–attenuated total reflection (FTIR–ATR) spectroscopy. The secondary structures of both model antigens change when adsorbed to aluminium hydroxide. The structural perturbation depends on the amount of adsorbed protein. Maximal adsorption gives a more native-like structure. This may indicate that protein is adsorbed in different manners depending on the concentration. The adsorbed antigens are released using phosphate buffer pH 7.4 (PB). The recovery is approximate 80% after 40 min in the presence of PB. The recovery curves of both proteins also indicate two different adsorption modes. FTIR–ATR and circular dichroism (CD) spectroscopy yield similar results suggesting that the adsorbed antigens refold to their native-like state after release.

Download Full-text

Phosphorylated human galectin-3: Facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2008.01.018 ◽

2008 ◽

Vol 1780 (4) ◽

pp. 716-722 ◽

Cited By ~ 32

Author(s):

Dieter Kübler ◽

Chien-Wen Hung ◽

Tarun K. Dam ◽

Jürgen Kopitz ◽

Sabine André ◽

...

Keyword(s):

Large Scale ◽

Structural Changes ◽

Cd Spectroscopy ◽

Large Scale Preparation ◽

Galectin 3 ◽

Scale Preparation

Download Full-text

Tumor protein D54 binds intracellular nanovesicles via an amphipathic lipid packing sensor (ALPS) motif

10.1101/2021.12.03.471088 ◽

2021 ◽

Author(s):

Antoine Reynaud ◽

Maud Magdeleine ◽

Amanda Patel ◽

Anne Sophie Gay ◽

Delphine Debayle ◽

...

Keyword(s):

Lipid Membranes ◽

Structural Changes ◽

Covalent Binding ◽

Limited Proteolysis ◽

Coiled Coil ◽

Tryptophan Fluorescence ◽

Cd Spectroscopy ◽

Dependent Manner ◽

Lipid Packing ◽

Physicochemical Features

AbstractTumor Protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54 and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical transport vesicles (e.g. COPI and COPII). Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by several amphipathic helices, which could fold upon binding to lipid membranes. One of these helices has the physicochemical features of an Amphipathic Lipid Packing Sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Limited proteolysis, CD spectroscopy, tryptophan fluorescence and cysteine mutagenesis coupled to covalent binding of a membrane sensitive probe show that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. TPD54 binding to artificial liposomes is very sensitive to liposome size and to lipid unsaturation but is poorly dependent on lipid charge. Cellular investigations confirmed the key role of the ALPS motif in vesicle targeting. Surprisingly, the vesicles selected by TPD54 poorly overlap with those captured by the golgin GMAP-210, a long vesicle tether at the Golgi apparatus, which displays a dimeric coiled-coil architecture and an N-terminal ALPS motif. We propose that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and -independent mechanisms.

Download Full-text

Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties

10.20944/preprints202012.0243.v1 ◽

2020 ◽

Author(s):

Darya Novopashina ◽

Mariya A. Vorobyeva ◽

Alexander A. Lomzov ◽

Vladimir N. Silnikov ◽

Alya G. Venyaminova

Keyword(s):

Molecular Biology ◽

Structural Changes ◽

Click Reaction ◽

Double Helix ◽

Chemical Properties ◽

Cd Spectroscopy ◽

Boron Clusters ◽

Strong Stabilization ◽

Duplex Stability ◽

Oligonucleotide Conjugates

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, closo-dodecaborate anion represents a high-boron containing residue with remarkable chemical stability and low toxicity, suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, while analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized DNA/DNA duplex but gave a strong stabilization of the duplex with RNA target. According to CD spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.

Download Full-text