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1.
Chem Sci ; 14(48): 14316-14326, 2023 Dec 13.
Article in English | MEDLINE | ID: mdl-38098704

ABSTRACT

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes. A strategy combining computational tools with experiments has shown that the substrate scope and enzymatic activity can be enhanced by rational engineering of certain important channels such as the substrate entry and water channels along with the active site of the enzyme. The evolved enzymes showed an improved catalytic rate for hexadecane hydroxylation with high regioselectivity. The Q67L/Y68F mutation showed binding of the substrate in the active site, water channel mutation L80F/V220T showed improved catalytic activity through the peroxide shunt pathway and substrate entry channel mutation W269F/I270A showed better substrate accessibility to the active pocket. All-atom MD simulations provided the rationale for the inactivity of the wild-type CYP175A1 for hexadecane hydroxylation and predicted the above hot-spot residues to enhance the activity. The reaction mechanism was studied by QM/MM calculations for enzyme-substrate complexes and reaction intermediates. Detailed thermal and thermodynamic stability of all the mutants were analyzed and the results showed that the evolved enzymes were thermally stable. The present strategy showed promising results, and insights gained from this work can be applied to the general enzymatic system to expand substrate scope and improve catalytic activity.

2.
J Environ Manage ; 328: 116978, 2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36521220

ABSTRACT

Growing industrialization and unchecked release of industrial waste, including heavy metals have resulted in disastrous effects on environment. Considering the problem of heavy metal pollution, the present research was designed to study the bioremediation of chromium, a highly toxic and prominent heavy metal pollutant by Acinetobacter junii strain b2w isolated from the Mithi river, Mumbai, India. The bacterial isolate could grow without affecting its growth kinetics up to a concentration of 200 ppm of chromium and showed resistance towards 400 ppm of chromium. It was able to bioremediate 83.06% of total chromium and reduces 98.24% of Cr6+ to C3+ at a concentration of 10 ppm of chromium. The bacterial isolate could grow well at a wide pH range from 5 to 9, salinity of up to 3.5% and could also tolerate heavy metals such as Cd, Zn, As, Hg, Pb and Cu. Thus, indicating its possible on-ground applicability for bioremediation of chromium. Acinetobacter junii bioaccumulate chromium without disrupting the cell integrity and biosorption. However, chromium alters the functional groups on bacterial cell surface and led to decrease in sulfate-containing molecules. Further, the protein expression study has revealed that Cr significantly up-regulates proteins broadly classified under envelope stress responses, oxidative stress responses, energy metabolism and quorum sensing and growth regulator. The possible mechanisms of Cr detoxification in Acinetobacter junii strain b2w could be reduction, bioaccumulation and efflux along with neutralization of oxidative stress generated by Cr. Thus, based on bacterial bioremediation potential and its molecular response, it can be proposed that the isolated Acinetobacter junii has potential applicability for chromium bioremediation.


Subject(s)
Chromium , Metals, Heavy , Biodegradation, Environmental , Proteomics , Metals, Heavy/analysis
3.
Appl Biochem Biotechnol ; 195(5): 3067-3095, 2023 May.
Article in English | MEDLINE | ID: mdl-36520354

ABSTRACT

The role of the shape of the nanostructure on the antibacterial effects of ZnO nanodisks has been investigated by detailed mass spectrometry-based proteomics along with other spectroscopic and microscopic studies on E. coli. The primary interaction study of the E. coli cells in the presence of ZnO nanodisks showed rigorous cell surface damage disrupting the cell wall/membrane components detected by microscopic and ATR-FTIR studies. Protein profiling of whole-cell extracts in the presence and absence of ZnO nanodisks identified several proteins that are upregulated and downregulated under the stress of the nanodisks. This suggests that the bacterial response to the primary stress leads to a secondary impact of ZnO nanodisk toxicity via regulation of the expression of specific proteins. Results showed that the ZnO nanodisks lead to the over-expression of peptidyl-dipeptidase Dcp, Transketolase-1, etc., which are important to maintaining the osmotic balance in the cell. The abrupt change in osmotic pressure leads to mechanical injury to the membrane, and nutritional starvation conditions, which is revealed from the expression of the key proteins involved in membrane-protein assembly, maintaining membrane integrity, cell division processes, etc. Thus, indicating a deleterious effect of ZnO nanodisk on the protective layer of E. coli. ZnO nanodisks seem to primarily affect the protective membrane layer, inducing cell death via the development of osmotic shock conditions, as one of the possible reasons for cell death. These results unravel a unique behavior of the disk-shaped ZnO nanostructure in executing lethality in E. coli, which has not been reported for other known shapes or morphologies of ZnO nanoforms.


Subject(s)
Nanostructures , Zinc Oxide , Escherichia coli , Zinc Oxide/pharmacology , Zinc Oxide/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Nanostructures/chemistry , Cell Membrane
4.
Protein J ; 41(6): 659-670, 2022 12.
Article in English | MEDLINE | ID: mdl-36273043

ABSTRACT

Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding temperature (Tm) of 88 °C (361 K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the effects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specificity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding affinity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufficient changes in the substrate binding pocket of the enzyme that can efficiently change substrate specificity of the enzyme.


Subject(s)
Cytochrome P-450 Enzyme System , Tyrosine , Substrate Specificity , Tyrosine/genetics , Molecular Docking Simulation , Cytochrome P-450 Enzyme System/chemistry , Fatty Acids , Mutation , Myristic Acids
8.
Angew Chem Int Ed Engl ; 61(13): e202116623, 2022 03 21.
Article in English | MEDLINE | ID: mdl-35005820

ABSTRACT

The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5-fold on mutation of Arg52 by His compared to the wild-type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir-embedded ferritin cages remained as stable as the wild-type analogue. These hybrid bio-nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming the corresponding chiral alcohols with up to 88 % conversion and 70 % enantioselectivity. An electron-withdrawing substituent on the reactant enhanced the Turnover frequency of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.


Subject(s)
Ferritins , Iridium , Catalysis , Ferritins/chemistry , Ferritins/genetics , Hydrogenation , Iridium/chemistry , Molecular Docking Simulation , Stereoisomerism
9.
Anal Biochem ; 626: 114204, 2021 08 01.
Article in English | MEDLINE | ID: mdl-33961907

ABSTRACT

Covalent linkage between the single-walled carbon nanotube (SWCNT) and CYP101 through a specific site of the enzyme can provide a novel method of designing efficient enzyme electrodes using this prototype cytochrome P450 enzyme. We have chemically modified the SWCNT with linker 4-carboxy phenyl maleimide (CPMI) containing maleimide functional groups. The enzyme was covalently attached on to the SWCNT through the maleimide group of the linker (CPMI) to the thiolate group of the surface exposed Cys 58 or Cys 136 of the CYP101 forming a covalently immobilized protein on the nanotube. Thin film of the modified SWCNT-CPMI-CYP101conjugate was made on a glassy carbon (GC) electrode. Direct electrochemistry of the substrate (camphor)-bound enzyme was studied using this immobilized enzyme electrode system and the redox potential was found to be -320mV vs Ag/AgCl (3 M KCl), which agrees with the redox potential of the substrate bound enzyme reported earlier. The electrochemically driven enzymatic mono-oxygenation of camphor by this immobilized enzyme electrode system was studied by measurement of the catalytic current at different concentrations of camphor. The catalytic current was found to increase with increasing concentration of camphor in presence of oxygen. The product formed during the catalysis was identified by mass-spectrometry as hydroxy-camphor.


Subject(s)
Biosensing Techniques/methods , Camphor 5-Monooxygenase/chemistry , Electrochemistry , Enzymes, Immobilized/chemistry , Mutation , Nanotubes, Carbon/chemistry , Camphor 5-Monooxygenase/genetics , Camphor 5-Monooxygenase/metabolism , Catalysis , Enzymes, Immobilized/metabolism , Humans
10.
J Biol Inorg Chem ; 26(4): 411-425, 2021 06.
Article in English | MEDLINE | ID: mdl-33928437

ABSTRACT

The role of the pentapeptide, NHSFM, derived from the surface exposed part of the metal ion binding loop of the subunit II of cytochrome c oxidase on the maturation of the binuclear purple CuA center of the enzyme has been investigated using several experimental and computational methods. The copper ion was found to form 1:1 complex of the pentapeptide with a binding constant ~ 104 M-1 to 105 M-1, where a 4 ligand coordination from the peptide in a type 2 copper center was revealed. The pH dependence of the metal-peptide was associated with a [Formula: see text] of ~ 10 suggesting deprotonation of the N-terminal amine. EXAFS studies as well as DFT calculations of the metal-peptide complexes revealed pH dependent changes in the metal-ligand bond distances. Spectroscopic properties of the metal peptides calculated from TDDFT studies agreed with the experimental results. Restrained molecular dynamics (RMD) simulations indicated coordination of a carbonyl oxygen from the asparagine (N) side chain and of water molecules apart from histidine (H), methionine (M) and terminal amine of asparagine (N) in a distorted square planar geometry of Cu-NHSFM. Analyses of the backbone distances as well as B-factors for the metal peptide suggested that the peptide backbone becomes more compact and rigid on binding of the metal ion. This indicated that binding of copper ion to this pentapeptide in the protein possibly cause movement of the protein backbone bringing other coordinating residues closer to the copper ion, and thus helping in sequential uptake of copper ions to the protein.


Subject(s)
Copper/chemistry , Electron Transport Complex IV/metabolism , Catalytic Domain , Copper/metabolism , Electron Transport Complex IV/genetics , Hydrogen-Ion Concentration , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Protein Subunits
11.
Dalton Trans ; 50(14): 4848-4858, 2021 Apr 14.
Article in English | MEDLINE | ID: mdl-33877182

ABSTRACT

Designing metal complexes as functional models for metalloenzymes remains one of the main targets in synthetic bioinorganic chemistry. Furthermore, the utilization of the product(s) derived from the catalytic reaction for subsequent organic transformation that occurs in biological systems is an even more difficult challenge for biochemists. Urease, the most efficient enzyme known, catalyzes the hydrolysis of urea and it contains an essential dinuclear NiII cluster in the active site. Inspired by the catalytic properties of urease, two dinickel(ii) complexes viz. Ni2L12(OAc)2(H2O) (1) and Ni2L22(OAc)2(H2O) (2) [HL1 = 2,4-dimethyl-6-{[(2'-dimethyl aminoethyl)methylamino]methyl}-phenol and HL2 = 2,4-dichloro-6-{[(2'-dimethyl aminoethyl)methylamino]methyl}-phenol] have been synthesized and characterized in this report. Both the complexes have shown the urease kind of activity with the liberation of ammonia from urea in aqueous solution. The plausible mechanistic pathway and kinetics of the reactions have been studied. Besides, the liberated ammonia has been utilized in the one-pot synthesis of biologically active products like 2-amino-3-cyanopyridines and their derivatives in aqueous medium with excellent yields.


Subject(s)
Coordination Complexes/chemistry , Nickel/chemistry , Pyridines/chemistry , Catalysis , Coordination Complexes/chemical synthesis , Kinetics , Molecular Conformation
12.
Biochem Biophys Res Commun ; 522(2): 506-511, 2020 02 05.
Article in English | MEDLINE | ID: mdl-31784087

ABSTRACT

The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Protein Multimerization , Animals , Drosophila Proteins/chemistry , Fluorescence Resonance Energy Transfer , Mutation, Missense/genetics , Protein Domains , Protein Subunits/metabolism
13.
Biochem Biophys Res Commun ; 518(1): 171-177, 2019 10 08.
Article in English | MEDLINE | ID: mdl-31420166

ABSTRACT

Association between two motor subunits through the rod/stalk domain enables molecular motors to walk processively on protein filaments. Previous studies suggested that structural flexibility in the coiled-coil stalk of kinesins is essential for processive runs. The stalk of heterotrimeric kinesin-2, a comparatively less processive motor, is unstable at ambient temperature. How this structural instability impacts the motor function is unclear. Here, using the Förster Resonance Energy Transfer based assays, we show that the Drosophila kinesin-2α/ß stalk heterodimer is dynamic at physiological conditions. We further show that insertion of a missense mutation (Glu551-Lys) at the C-terminal half of kinesin-2α stalk reduces the dynamics of the heterodimeric stalk in vitro. The mutation, isolated as a recessive lethal allele in a forward genetic screen, is reported to disrupt the motor function in axonal transport and cilia development. Together these two results suggest that the dynamic instability of the kinesin-2 stalk could play a crucial role in maintaining its biological function.


Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Kinesins/chemistry , Kinesins/metabolism , Protein Multimerization , Amino Acid Sequence , Animals , Fluorescence Resonance Energy Transfer , Mutation, Missense/genetics , Protein Domains , Protein Stability , Protein Subunits/chemistry , Protein Subunits/metabolism , Structure-Activity Relationship
14.
Dalton Trans ; 45(44): 17624-17632, 2016 Nov 28.
Article in English | MEDLINE | ID: mdl-27747364

ABSTRACT

Three stable copper complexes of peptides derived from the copper ion binding loop of the subunit II of cytochrome c oxidase have been prepared and characterized by various spectroscopic techniques. These stable copper complexes of peptides were found to exhibit cysteine, histidine and/or methionine ligation, which has predominant σ-contribution in the Cys-Cu charge transfer. The copper(ii) peptide complexes showed type-2 EPR spectra, which is uncommon in copper-cysteinate complexes. UV-visible spectra, Raman and EPR results support a tetragonal structure of the coordination geometry around the copper ion. The copper complex of the 9-amino acid peptide suggested the formation of a 'red' copper center while the copper complexes of the 12- and 11-amino acid peptides showed the formation of a 'green' copper center. The results provide insights on the first stable models of the copper complexes formed in the peptide scaffold that mimic the mono-nuclear copper bound protein intermediates proposed during the formation of the binuclear Cu2S2 core of the enzyme. These three copper complexes of peptides derived from the metal ion binding loop of the CuA center of the subunit II of cytochrome c oxidase showed novel spectroscopic properties which have not so far been reported in any stable small complex.


Subject(s)
Biomimetic Materials/chemistry , Coordination Complexes/chemistry , Copper/chemistry , Electron Transport Complex IV/chemistry , Peptides/chemistry , Amino Acid Sequence , Animals , Cattle , Electron Spin Resonance Spectroscopy , Humans , Models, Molecular , Protein Subunits/chemistry , Rats , Thermus/chemistry , Thermus/enzymology
15.
Biochem Biophys Res Commun ; 472(1): 189-93, 2016 Mar 25.
Article in English | MEDLINE | ID: mdl-26923072

ABSTRACT

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.


Subject(s)
Cytoplasmic Dyneins/metabolism , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide/biosynthesis , Calmodulin/metabolism , Cytoplasmic Dyneins/genetics , Electron Transport , Enzyme Inhibitors/metabolism , Heme/metabolism , Humans , In Vitro Techniques , Kinetics , NADP/metabolism , Nitric Oxide Synthase Type I/genetics , Oxidation-Reduction , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
16.
Bioorg Chem ; 62: 94-105, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26312734

ABSTRACT

The thermostable nature of CYP175A1 enzyme is of potential interest for the biocatalysis at ambient temperature or at elevated temperature under environmentally benign conditions. Although little is known about the substrate selectivity of this enzyme, the biocatalytic activities of CYP175A1 on different substituted naphthalenes have been studied in oxidative pathway, and the effect of the substituent on the reaction has been determined. The enzyme first acts as a peroxygenase to convert these substituted naphthalenes to the corresponding naphthols, which subsequently undergo in-situ oxidative dimerization to form dyes of different colors possibly by the peroxidase-type activity of CYP175A1. The product analyses and kinetic measurements suggested that the presence of electron releasing substituent (ERS) in the substrate enhanced the substrate conversion, whereas the presence of electron withdrawing substituent (EWS) in the substrate drastically reduced the substrate conversion. The position of the ERS in the substrate was also found to play an important role in the transformation of the substrate. The results further demonstrate that mutation of the Leu80 residue to Phe enhances the reactivity of the enzyme by favoring the substrate association in the active site. The observed rates of the enzymatic oxygenation reaction of the substituted naphthalenes followed the Hammett correlation of substituent effect, supporting aromatic electrophilic substitution mechanism catalyzed by the cytochrome P450 enzyme.


Subject(s)
Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Naphthalenes/chemistry , Bacterial Proteins/genetics , Cytochrome P-450 Enzyme System/genetics , Enzyme Assays , Naphthalenes/metabolism , Spectrophotometry
17.
Chem Commun (Camb) ; 51(83): 15257-60, 2015 Oct 25.
Article in English | MEDLINE | ID: mdl-26176025

ABSTRACT

We report the use of a molecular peroxidase mimic biuret-Fe-TAML for chemoselective labeling of proteins and the subsequent visual detection (<0.1 pmoles) of the conjugate in a polyacrylamide gel by catalytic signal amplification. Use of this probe in activity based protein profiling (ABPP) of serine proteases is also demonstrated.


Subject(s)
Acrylic Resins/chemistry , Acrylic Resins/metabolism , Iron Compounds/chemistry , Proteins/analysis , Animals , Catalysis , Cattle , Gels/chemistry , Gels/metabolism , Humans , Iron Compounds/metabolism , Models, Molecular , Molecular Structure , Proteins/metabolism
18.
Biochemistry ; 52(27): 4620-35, 2013 Jul 09.
Article in English | MEDLINE | ID: mdl-23745508

ABSTRACT

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.


Subject(s)
Copper/chemistry , Electron Transport Complex IV/chemistry , Thermus thermophilus/enzymology , Base Sequence , Circular Dichroism , DNA Primers , Electron Spin Resonance Spectroscopy , Electron Transport Complex IV/genetics , Kinetics , Mutagenesis, Site-Directed , Spectrophotometry, Ultraviolet
19.
Biochemistry ; 52(8): 1373-84, 2013 Feb 26.
Article in English | MEDLINE | ID: mdl-23339332

ABSTRACT

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.


Subject(s)
Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Protein Stability , Protein Unfolding , Thermus thermophilus/enzymology , Thermus thermophilus/genetics , Catalytic Domain/drug effects , Circular Dichroism , Cytochrome c Group/metabolism , Electrochemical Techniques , Guanidine/pharmacology , Heme/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Point Mutation , Protein Conformation/drug effects , Protein Stability/drug effects , Protein Unfolding/drug effects , Spectrophotometry, Ultraviolet , Temperature , Thermodynamics , Thermus thermophilus/chemistry , Thermus thermophilus/metabolism
20.
PLoS One ; 7(9): e45981, 2012.
Article in English | MEDLINE | ID: mdl-23029351

ABSTRACT

DEFINITION: Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors. BACKGROUND: Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature. CONCLUSIONS/SIGNIFICANCE: Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated.


Subject(s)
Drosophila Proteins/chemistry , Drosophila/chemistry , Kinesins/chemistry , Molecular Dynamics Simulation , Amino Acid Sequence , Animals , Circular Dichroism , Drosophila Proteins/ultrastructure , Kinesins/ultrastructure , Molecular Sequence Data , Protein Folding , Protein Multimerization , Protein Stability , Protein Structure, Secondary
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