Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

1977 ◽  
Vol 55 (1) ◽  
pp. 43-49 ◽  
Author(s):  
M. Kapoor ◽  
M. D. O'Brien
Keyword(s):  
Pyruvate Kinase ◽  
Functional Groups ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Complex Cross ◽  
Dimethyl Suberimidate ◽  
Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

Cross-linking of Hemoglobin, Haptoglobin, and Hemoglobin–Haptoglobin Complex with Bifunctional Imidoesters

1975 ◽  
Vol 53 (8) ◽  
pp. 861-867 ◽  
Author(s):  
W. L. Lockhart ◽  
David B. Smith
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spatial Relation ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Cross Link ◽  
Complex Cross ◽  
Cross Links

Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin–haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin–haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin.Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted.

scholarly journals Cross-linking reveals laminin coiled-coil architecture

2016 ◽  
Vol 113 (47) ◽  
pp. 13384-13389 ◽  
Author(s):  
Gad Armony ◽  
Etai Jacob ◽  
Toot Moran ◽  
Yishai Levin ◽  
Tevie Mehlman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Self Assembly ◽  
Quaternary Structure ◽  
Coiled Coil ◽  
Surface Proteins ◽  
Cross Linking ◽  
Single Particle Reconstruction ◽  
Cross Links ◽  
Particle Reconstruction ◽  
Key Questions

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.

scholarly journals Chemical modifications of metallothionein. Preparation and characterization of polymers

1984 ◽  
Vol 221 (3) ◽  
pp. 569-575 ◽  
Author(s):  
D M Templeton ◽  
M G Cherian
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Quaternary Structure ◽  
Biological Significance ◽  
Cross Linking ◽  
Metal Binding Sites ◽  
Polymeric Species ◽  
Lysine Residues ◽  
Dimethyl Suberimidate ◽  
High Yields

Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.

scholarly journals Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality

2020 ◽  
Vol 477 (12) ◽  
pp. 2295-2312
Author(s):  
Rafael Medina ◽  
Deisy Perdomo ◽  
Carolina Möller ◽  
José Bubis
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Bovine Rhodopsin ◽  
Rhodopsin Kinase ◽  
Cross Links ◽  
Guanine Nucleotide Binding

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

scholarly journals Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Carla Schmidt ◽  
Min Zhou ◽  
Hazel Marriott ◽  
Nina Morgner ◽  
Argyris Politis ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Mass Spectra ◽  
Nucleotide Binding ◽  
Cross Linking ◽  
Spinach Chloroplasts ◽  
Cross Links ◽  
Chemical Cross Linking

Abstract F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid ‘plug’ in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

Use of a Diimidoester Cross-Linking Reagent to Examine the Submit Structure of Rabbit Muscle Pyruvate Kinase

1972 ◽  
Vol 50 (4) ◽  
pp. 416-422 ◽  
Author(s):  
Gregg E. DaVies ◽  
J. Gordin Kaplan
Keyword(s):  
Pyruvate Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Rabbit Muscle ◽  
Cross Links ◽  
Primary Amino ◽  
Muscle Pyruvate Kinase ◽  
Dimeric Intermediate ◽  
Hydrolysis Of

Rabbit muscle pyruvate kinase, a tetramer of highly similar or identical subunits, is known to dissociate in urea via a dimeric intermediate. To provide additional evidence regarding the structure of the native oligomer, we have treated pyruvate kinase with dimethyl pimelimidate and examined the yields of cross-linked species resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Of four products resolved, predominating species are dimers and tetramers of the pyruvate kinase subunit, while monomers and trimers are present in lesser amounts. These relative yields are consistent with a dimeric structure of the tetramer.In a typical cross-linking reaction, about 86 of the 148 primary amino groups in the pyruvate kinase tetramer are amidinated. Of 53 moles of cross-linking reagent incorporated per mole of tetramer, 38% have reacted monofunctionally, with hydrolysis of the second imidoester group, and 62% have reacted bifunctionally to form intra- and intersubunit cross-links.

scholarly journals Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

2016 ◽  
Vol 1 ◽  
pp. 5 ◽  
Author(s):  
Zhuo Chen ◽  
Lutz Fischer ◽  
Salman Tahir ◽  
Jimi-Carlo Bukowski-Wills ◽  
Paul Barlow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Complement C3 ◽  
Cross Linking ◽  
Complement Protein ◽  
Protein Protein Interaction ◽  
Code Of Practice ◽  
Final Maturation ◽  
Cross Links ◽  
Binary Switching

Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.

scholarly journals Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking

2010 ◽  
Vol 430 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Zhanjun Hou ◽  
Jianmei Wu ◽  
Jun Ye ◽  
Christina Cherian ◽  
Larry H. Matherly
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Specific Binding ◽  
Substrate Binding ◽  
Residual Activity ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Reduced Folate Carrier ◽  
Cross Links ◽  
Hydrophilic Loop

RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6 and N6–N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.

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