RNase A – tRNA binding alters protein conformation

2007 ◽  
Vol 85 (3) ◽  
pp. 311-318 ◽  
Author(s):  
C.N. N’soukpoé-Kossi ◽  
C. Ragi ◽  
H.A. Tajmir-Riahi
Keyword(s):  
Binding Sites ◽  
Binding Constant ◽  
Structural Changes ◽  
Structural Information ◽  
Binding Mode ◽  
Rnase A ◽  
Random Coil ◽  
Pancreatic Ribonuclease A ◽  
Α Helix ◽  
Rna Interaction

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5′ bonds in RNA on the 3′ side of pyrimidine to form cyclic 2′,5′-phosphates. Even though extensive structural information is available on RNase A complexes with mononucleotides and oligonucleotides, the interaction of RNase A with tRNA has not been fully investigated. We report the complexation of tRNA with RNase A in aqueous solution under physiological conditions, using a constant RNA concentration and various amounts of RNase A. Fourier transform infrared, UV-visible, and circular dichroism spectroscopic methods were used to determine the RNase binding mode, binding constant, sequence preference, and biopolymer secondary structural changes in the RNase–tRNA complexes. Spectroscopic results showed 2 major binding sites for RNase A on tRNA, with an overall binding constant of K = 4.0 × 105 (mol/L)–1. The 2 binding sites were located at the G-C base pairs and the backbone PO2 group. Protein–RNA interaction alters RNase secondary structure, with a major reduction in α helix and β sheets and an increase in the turn and random coil structures, while tRNA remains in the A conformation upon protein interaction. No tRNA digestion was observed upon RNase A complexation.

The effect of aspirin-HSA complexation on the protein secondary structure

2000 ◽  
Vol 78 (2) ◽  
pp. 291-296 ◽  
Author(s):  
J F Neault ◽  
A Novetta-Delen ◽  
H Arakawa ◽  
H Malonga ◽  
H A Tajmir-Riahi
Keyword(s):  
Secondary Structure ◽  
Binding Constant ◽  
Structural Changes ◽  
Resolution Enhancement ◽  
Protein Secondary Structure ◽  
Binding Mode ◽  
Drug Binding ◽  
Drug Concentrations ◽  
Fitting Procedures ◽  
Α Helix

This study was designed to determine the secondary structure of human serum albumin (HSA) in the presence of aspirin in H2O and D2O solutions at physiological pH, using aspirin concentrations of 0.0001-5 mM with final protein concentration of 2% w/v. UV-vis spectra and Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution, second derivative resolution enhancement, and curve-fitting procedures were applied to characterize the drug binding mode, the binding constant, and the protein secondary structure in the aspirin-HSA complexes. Spectroscopic evidence showed that no aspirin-protein interaction occurs at very low drug concentration (0.0001 mM), whereas at higher drug contents (0.001-0.1 mM) the aspirin anion binding (H-bonding) is mainly through the ε-amino NH3+ group with overall binding constant of K = 1.4 × 104 M-1. At high drug concentrations (1-5 mM), acetylation of Lys-199 was observed. Aspirin binding results in protein secondary structural changes from that of the α-helix 55% (free HSA) to 49%, β-sheet 22% (free HSA) to 31%, β-anti 12% (free HSA) to 4% and turn 11% (free HSA) to 16% in the aspirin-HSA complexes..Key words: aspirin, protein, drug, binding mode, binding constant secondary structure, FTIR spectroscopy.

Structural Changes of Almost Completely Inactivated Papain with Higher Concentration of Parachloromercuribenzoic Acid

2019 ◽  
Vol 11 (5) ◽  
pp. 704-710
Author(s):  
Xueying Liu ◽  
Xiaoli Cao ◽  
Min Li ◽  
Yinyan Chen ◽  
Xiaojuan Li ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Structural Changes ◽  
Binding Pocket ◽  
Enzyme Engineering ◽  
Attenuated Total Reflection ◽  
Random Coil ◽  
Inhibition Mechanism ◽  
Hydrophobic Force ◽  
Key Functional Groups ◽  
Α Helix

A hydrolysis of casein by papain was carried out to explore interaction between parachloromercuribenzoic acid (PCMB) and papain through Attenuated Total Reflection Flourier transformed Infrared Spectroscopy (ATR-FTIR), Trp intrinsic fluorescence spectroscopy and molecular docking analysis. Papain activity was almost completely inactivated when PCMB concentration reached to 10–4 mol/L or higher. The content of α-helix was decreased from 36.79% to 19.79% even up to 17%. On the other hand, the content of β-sheet was decreased from 13.41% to 11.57% by a little degree of 1.84%. However, the contents of β-turn, random coil and intermolecular β-sheet aggregates were elevated by different degree of 2.67%, 8.87% and 7.3%, respectively at the papain exposure of 10–4 mol/L PCMB solution. PCMB could bind to papain mainly through hydrophobic force and hydrogen bonding. The hydrogen bond between PCMB and Gly66; while, the hydrophobic bonds between PCMB and Trp26 in the ligand-binding pocket of papain might be the key functional groups for the inhibition of papain activity. The L-domain of papain was destructed, leading to the inactivation and fluorescence quenching of papain. The inhibition mechanism of papain activity by 10–4mol/L PCMB was involved the formation of hydrophobic force and hydrogen bond between PCMB and papain. This attempt successfully brought forth the application of computational approaches to dissect the inhibition mechanism and directed design in enzyme engineering area.

Laue and monochromatic crystallography on carbonic anhydrase

1992 ◽  
Vol 340 (1657) ◽  
pp. 301-309 ◽  
Keyword(s):  
Carbonic Anhydrase ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Binding Mode ◽  
Molecular Structures ◽  
Data Sets ◽  
Time Resolved ◽  
Zinc Enzyme ◽  
Unexpected Findings

We wanted to analyse the binding mode of small anion inhibitors to the zinc enzyme carbonic anhydrase in order to explore the binding of substrates and the catalytic mechanism of the enzyme. This was started by recording two data-sets by Laue diffraction to obtain the wanted structural information. In addition we wanted to test the capacity of the Laue method to show the small structural changes that are often associated with the catalytic activity of m any enzymes. To be able to exploit fully time-resolved crystallography the method should be able to detect such minor structural changes. The obtained Laue results did not agree with the expected molecular structures. Thus we needed to record monochromatic data-sets of the same states of the enzyme to confirm our results. All major findings from the Laue data agree with the monochromatic data. Stimulated by the unexpected findings we have continued the investigations of anion binding to carbonic anhydrase. We have studied both the zinc enzyme and replaced the native metal by cobalt which also yields an active enzyme. The accumulated picture of the ligand binding to the enzyme sheds new light on the substrate binding and on the catalytic mechanism.

scholarly journals Differential BUM-HMM: a robust statistical modelling approach for detecting RNA flexibility changes in high-throughput structure probing data

2020 ◽  
Author(s):  
Paolo Marangio ◽  
Ka Ying Toby Law ◽  
Guido Sanguinetti ◽  
Sander Granneman
Keyword(s):  
Protein Binding ◽  
High Throughput ◽  
Conformational Changes ◽  
Binding Sites ◽  
Rna Structure ◽  
Structural Changes ◽  
High Throughput Sequencing ◽  
Structural Information ◽  
Protein Binding Sites ◽  
Structure Probing

Combining RNA structure probing with high-throughput sequencing technologies has greatly enhanced our ability to analyze RNA structure at transcriptome scale. However, the high level of noise and variability encountered in these data call for the development of computational tools that robustly extract RNA structural information. Here we present diffBUM-HMM, a noise-aware model that enables accurate detection of RNA flexibility and conformational changes from high-throughput RNA structure-probing data. DiffBUM-HMM is compatible with a wide variety of high-throughput RNA structure probing data, taking into consideration biological variation, sequence coverage and sequence representation biases. We demonstrate that, compared to the existing approaches, diffBUM-HMM displays higher sensitivity while calling virtually no false positives. DiffBUM-HMM analysis of ex vivo and in vivo Xist SHAPE-MaP data detected many more RNA structural differences, involving mostly single-stranded nucleotides located at or near protein-binding sites. Collectively, our analyses demonstrate the value of diffBUM-HMM for quantitatively detecting RNA structural changes and reinforce the notion that RNA structure probing is a very powerful tool for identifying protein-binding sites.

DNase I – DNA interaction alters DNA and protein conformations

2008 ◽  
Vol 86 (3) ◽  
pp. 244-250 ◽  
Author(s):  
C.N. N’soukpoé-Kossi ◽  
S. Diamantoglou ◽  
H.A. Tajmir-Riahi
Keyword(s):  
Binding Constant ◽  
Protein Secondary Structure ◽  
Binding Mode ◽  
Dna Interaction ◽  
Minor Groove ◽  
Dnase I ◽  
Protein Conformations ◽  
Physiological Conditions ◽  
Α Helix ◽  
The Right

Human DNase I is an endonuclease that catalyzes the hydrolysis of double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions in the presence of divalent Mg and Ca cations. It binds to the minor groove and the backbone phosphate group and has no contact with the major groove of the right-handed DNA duplex. The aim of this study was to examine the effects of DNase I – DNA complexation on DNA and protein conformations.We monitored the interaction of DNA with DNase I under physiological conditions in the absence of Mg2+, with a constant DNA concentration (12.5 mmol/L; phosphate) and various protein concentrations (10–250 µmol/L). We used Fourier transfrom infrared, UV-visible, and circular dichroism spectroscopic methods to determine the protein binding mode, binding constant, and effects of polynucleotide–enzyme interactions on both DNA and protein conformations. Structural analyses showed major DNase–PO2 binding and minor groove interaction, with an overall binding constant, K, of 5.7 × 105 ± 0.78 × 105 (mol/L)–1. We found that the DNase I – DNA interaction altered protein secondary structure, with a major reduction in α helix and an increase in β sheet and random structures, and that a partial B-to-A DNA conformational change occurred. No DNA digestion was observed upon protein–DNA complexation.

scholarly journals Structure of the carboxy-terminal domain ofMycobacterium tuberculosisCarD protein: an essential rRNA transcriptional regulator

2014 ◽  
Vol 70 (2) ◽  
pp. 160-165 ◽  
Author(s):  
Shanti P. Gangwar ◽  
Sita R. Meena ◽  
Ajay K. Saxena
Keyword(s):  
Rna Polymerase ◽  
Structural Changes ◽  
Genotoxic Stress ◽  
Random Coil ◽  
Β Subunit ◽  
Thermus Aquaticus ◽  
Terminal Domain ◽  
Secondary Structure Analysis ◽  
Α Helix ◽  
Carboxy Terminal

The CarD protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. The CarD protein binds the β subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the β subunit of RNA polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. To understand the structure of CarD and its interaction with the β subunit of RNA polymerase,Mycobacterium tuberculosisCarD (MtbCarD) and theThermus aquaticusRNA polymerase β subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated thatMtbCarD contains ∼60% α-helix, ∼7% β-sheet and ∼33% random-coil structure. The C-terminal domain ofMtbCarD (CarD83–161) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD83–161forms a distorted Y-shaped structure containing bundles of three helices connected by a loop. The residues forming the distorted Y-shaped structure are highly conserved in CarD sequences from other mycobacterial species. Comparison of the CarD83–161structure with the recently determined full-lengthM. tuberculosisandT. thermophilusCarD crystal structures revealed structural differences in residues 141–161 of the C-terminal domain of the CarD83–161structure. The structural changes in the CarD83–161structure occurred owing to proteolysis and crystallization artifacts.

scholarly journals Structural analysis of protein–DNA and protein–RNA interactions by FTIR, UV-visible and CD spectroscopic methods

2009 ◽  
Vol 23 (2) ◽  
pp. 81-101 ◽  
Author(s):  
H. A. Tajmir-Riahi ◽  
C. N. N'soukpoé-Kossi ◽  
D. Joly
Keyword(s):  
Binding Sites ◽  
Ribonuclease A ◽  
Rnase A ◽  
Fundamental Question ◽  
Spectroscopic Methods ◽  
Deoxyribonuclease I ◽  
Calf Thymus ◽  
Uv Visible ◽  
Rna Interaction ◽  
Structural Aspects

In this chapter the fundamental question of how does protein–DNA or protein–RNA interaction affect the structures and dynamics of DNA, RNA and protein is addressed. Models for calf-thymus DNA and transfer RNA interactions with human serum albumin (HSA), ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) are presented here, using Fourier Transform Infrared (FTIR) spectroscopy in conjunction with UV-visible and CD spectroscopic methods. In the models considered, the binding sites, stability and structural aspects of protein–DNA and protein–RNA are discussed and the effects of protein interaction on the secondary structures of DNA, RNA and protein were determined.

scholarly journals A Spectroscopic Study on PtCl4(2−) Binding to Rabbit Skeletal Muscle G-Actin

1995 ◽  
Vol 2 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Juan Zou ◽  
Hong-Ye Sun ◽  
Kui Wang
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Binding Constant ◽  
Intrinsic Fluorescence ◽  
Molar Ratio ◽  
Second Step ◽  
High Affinity ◽  
Α Helix ◽  
Discontinuous Mode ◽  
Sulfur Containing

It was found that the binding of PtCl42− to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n1 = 30 with average binding constant K1=1.0×107M-1. The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl42−binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n2=26 with average binding constant K2=2.1×106M-1. Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl42− binding, the G-actin turns to an open and loose structure in a discontinuous mode.

scholarly journals Multiomics study of a heterotardigrade, Echinisicus testudo, suggests convergent evolution of anhydrobiosis-related proteins in Tardigrada

2020 ◽  
Author(s):  
Yumi Murai ◽  
Maho Yagi-Utsumi ◽  
Masayuki Fujiwara ◽  
Masaru Tomita ◽  
Koichi Kato ◽  
...  
Keyword(s):  
Water Content ◽  
Molecular Mechanism ◽  
Convergent Evolution ◽  
Structural Changes ◽  
Extreme Environments ◽  
Random Coil ◽  
Related Protein ◽  
Α Helix ◽  
Related Proteins

AbstractMany limno-terrestrial tardigrades can enter an ametabolic state upon desiccation, in which the animals can withstand extreme environments. To date, studies of the molecular mechanism have predominantly investigated the class Eutardigrada, and that in the Heterotardigrada, remains elusive. To this end, we report a multiomics study of the heterotardigrade Echiniscus testudo, which is one of the most desiccation-tolerant species. None of the previously identified tardigrade-specific anhydrobiosis-related genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families of novel abundant heat-soluble proteins and the proteins exhibited structural changes from random coil to α-helix as the water content decreased in vitro. These characteristics are analogous to those of anhydrobiosis-related protein in eutardigrades, while there is no conservation at the sequence level. Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada.

scholarly journals Structure and Dynamics of DNA-bound Ruthenium Complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1377-C1377
Author(s):  
James Hall ◽  
Kyra O'Sullivan ◽  
Hakan Niyazi ◽  
Juan Sanchez-Weatherby ◽  
Graeme Winter ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Photodynamic Therapy ◽  
Binding Sites ◽  
Structural Information ◽  
Ruthenium Complexes ◽  
Binding Mode ◽  
Sequence Specificity ◽  
Structure And Dynamics ◽  
Dna Oligonucleotides ◽  
High Level

Since the 1980s, there has been great interest in how polypyridyl ruthenium complexes bind to DNA. This is due to their photoactive properties[1], which have great potential in photodynamic therapy, as they are able to damage DNA upon photoirradiation. However, there has been significant debate over the precise binding sites of these complexes due to the lack of definitive structural information. Presented here are several high resolution crystal structures showing how these complexes can bind to short DNA oligonucleotides. With each new structure we are able to answer questions about the binding geometry and step specificity which could explain the observations obtained from biophysical measurements in solution. We have shown that the complexes bind by intercalation as well as confirming a previously proposed binding mode, semi-intercalation. We have also shown that the complexes bind with a high level of sequence specificity[2], preferring TA steps over AT and CG and that each enantiomer can bind with a different orientation[3] (Figure 1). One obvious advantage to working with crystal samples is that they possess a well defined molecular structure, which can be determined and is therefore known. Spectroscopic experiments, with data collected in the picosecond and nanosecond timescale, will also be reported with these systems.

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