pH and Temperature-Induced Molten Globule-Like Denatured States of Equinatoxin II:  A Study by UV-Melting, DSC, Far- and Near-UV CD Spectroscopy, and ANS Fluorescence†

Biochemistry ◽  
1997 ◽  
Vol 36 (47) ◽  
pp. 14345-14352 ◽  
Author(s):  
Nataša Poklar ◽  
Jurij Lah ◽  
Mateja Salobir ◽  
Peter Maček ◽  
Gorazd Vesnaver
Keyword(s):  
Molten Globule ◽  
Cd Spectroscopy ◽  
Equinatoxin Ii ◽  
Denatured States ◽  
Uv Melting

Models of cooperativity in protein folding

1995 ◽  
Vol 348 (1323) ◽  
pp. 61-70 ◽  
Keyword(s):  
Protein Folding ◽  
Molten Globule ◽  
Core Collapse ◽  
Hydrophobic Core ◽  
Model Studies ◽  
Helix Formation ◽  
Collapse Model ◽  
Folding Pathways ◽  
State Behaviour ◽  
Denatured States

What is the basis for the two-state cooperativity of protein folding? Since the 1950s, three main models have been put forward. 1. In ‘helix-coil’ theory, cooperativity is due to local interactions among near neighbours in the sequence. Helix-coil cooperativity is probably not the principal basis for the folding of globular proteins because it is not two-state, the forces are weak, it does not account for sheet proteins, and there is no evidence that helix formation precedes the formation of a hydrophobic core in the folding pathways. 2. In the ‘sidechain packing’ model, cooperativity is attributed to the jigsaw-puzzle-like complementary fits of sidechains. This too is probably not the basis of folding cooperativity because exact models and experiments on homopolymers with sidechains give no evidence that sidechain freezing is two-state, sidechain complementarities in proteins are only weak trends, and the molten globule model predicted by this model is far more native-like than experiments indicate. 3. In the ‘hydrophobic core collapse’ model, cooperativity is due to the assembly of non-polar residues into a good core. Exact model studies show that this model gives two-state behaviour for some sequences of hydrophobic and polar monomers. It is based on strong forces. There is considerable experimental evidence for the kinetics this model predicts: the development of hydrophobic clusters and cores is concurrent with secondary structure formation. It predicts compact denatured states with sizes and degrees of disorder that are in reasonable agreement with experiments.

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

2006 ◽  
Vol 84 (2) ◽  
pp. 126-134 ◽  
Author(s):  
Fouzia Rashid ◽  
Sandeep Sharma ◽  
M A Baig ◽  
Bilqees Bano
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Structural Features ◽  
Cd Spectroscopy ◽  
Native State ◽  
Molten Globule State ◽  
Fluorescence Measurements ◽  
Helical Content ◽  
Human Placental Cystatin

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of α-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.Key words: human placental cystatin, molten globule, acid-induced state, trifluoroethanol, methanol, CD spectroscopy, ANS fluorescence, pH, protein folding.

scholarly journals Effect of temperature on the secondary structure of β-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis

1997 ◽  
Vol 324 (1) ◽  
pp. 341-346 ◽  
Author(s):  
Xiao Lin QI ◽  
Carl HOLT ◽  
David MCNULTY ◽  
David T. CLARKE ◽  
Sharon BROWNLOW ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Molten Globule ◽  
Cd Spectroscopy ◽  
Helical Conformation ◽  
Effect Of Temperature ◽  
Sheet Structure ◽  
Good Signal ◽  
Increasing Temperature ◽  
Β Lactoglobulin ◽  
Β Sheet

Previous CD measurements of changes in the conformation of β-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 °C. New CD measurements are reported at temperatures up to 80 °C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 °C with a ZnSe circle cell and a single simplified model of the substructure of the amide I′ band was used to give the fractional contents of β-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of β-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 °C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 °C a partly unfolded state is formed, possibly by destabilization of the intermolecular β-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Studies on the Interaction of Papain with Human Placental Cystatin by Uv, Fluorescence and Cd Spectroscopy

2004 ◽  
Vol 11 (6) ◽  
pp. 583-591 ◽  
Author(s):  
Fouzia Rashid ◽  
S. Baba ◽  
Sandeep Sharma ◽  
B. Bano
Keyword(s):  
Cd Spectroscopy ◽  
Uv Fluorescence ◽  
Human Placental Cystatin

Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues.

2020 ◽  
Vol 27 ◽  
Author(s):  
Sheetal Uppal ◽  
Mohd. Asim Khan ◽  
Suman Kundu
Keyword(s):  
Molten Globule ◽  
Life Forms ◽  
Model System ◽  
Heme Pocket ◽  
The Past ◽  
Specific Manner ◽  
Delivery Vehicles ◽  
The Stability ◽  
Key Residues ◽  
Synechocystis Sp

Aims: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. Background: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as “Synechocystis hemoglobin (SynHb)”. The “three histidines” linkages to heme are novel to this cyanobacterial hemoglobin. Objective: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. Methods: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. Result: The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a “molten globule” like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. Conclusion: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

Cytotoxic Activity and DNA Binding Property of New Aminopyrimidine Derivatives

2020 ◽  
Vol 17 (5) ◽  
pp. 640-654
Author(s):  
Hamidreza Akrami ◽  
Bibi Fatemeh Mirjalili ◽  
Omidreza Firuzi ◽  
Azadeh Hekmat ◽  
Ali Akbar Saboury ◽  
...  
Keyword(s):  
Cell Lines ◽  
Anticancer Agents ◽  
Lymphoblastic Leukemia ◽  
Cd Spectroscopy ◽  
Myelogenous Leukemia ◽  
Breast Adenocarcinoma ◽  
Binding Property ◽  
Anticancer Compounds ◽  
Calf Thymus ◽  
Dna Binding Property

Background: Chromene and anilinopyrimidine heterocyclics are attractive anticancer compounds that have inspired many researchers to design novel derivatives bearing improved anticancer activity. Methods: A series of pyrimidine-fused benzo[f]chromene derivatives 6a-x were synthesized as anticancer hybrids of 1H-benzo[f]chromenes and anilinopyrimidines. The inhibitory activity of the synthesized compounds 6a-x against cell viability of human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (MOLT-4) and human breast adenocarcinoma (MCF-7) cell lines was evaluated using MTT assay. The interaction of the most promising compound with calf-thymus DNA was also studied using spectrometric titrations and Circular Dichroism (CD) spectroscopy. Results: Most compounds showed promising activity against tested cell lines. Among them, 2,4- dimethoxyanilino derivative 6g exhibited the best profile of activity against tested cell lines (IC50s = 1.6-6.1 μM) with no toxicity against NIH3T3 normal cell (IC50 >200 μM). The spectrometric studies exhibited that compound 6g binds to DNA strongly and may change DNA conformation significantly, presumably via a groove binding mechanism. Conclusion: The results of this study suggest that the prototype compound 6g can be considered as a novel lead compound for the design and discovery of novel anticancer agents.

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD)

2019 ◽  
Vol 15 (5) ◽  
pp. 486-495 ◽  
Author(s):  
Li-Xin Peng ◽  
Xue-Hui Liu ◽  
Bo Lu ◽  
Si-Ming Liao ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Polysialic Acid ◽  
Neuronal Cell ◽  
Cd Spectroscopy ◽  
Migration And Invasion ◽  
Heparin Binding ◽  
Clinical Prognosis ◽  
Quenching Analysis ◽  
Different Types ◽  
Α Helix

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

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