Chemical cross-linking and analytical ultracentrifugation study of the histone-like protein HBsu: Quaternary structure and DNA binding

2007 ◽  
pp. 74-81
Author(s):  
C. Timmermann ◽  
J. Behlke ◽  
O. Ristau ◽  
H. Gerst ◽  
U. Heinemann
Keyword(s):  
Dna Binding ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Cross Linking ◽  
Chemical Cross Linking

scholarly journals Proper evaluation of chemical cross-linking-based spatial restraints improves the precision of modeling homo-oligomeric protein complexes

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Aljaž Gaber ◽  
Gregor Gunčar ◽  
Miha Pavšič
Keyword(s):  
Quaternary Structure ◽  
Comprehensive Evaluation ◽  
Protein Complexes ◽  
Scoring Function ◽  
Cross Linking ◽  
High Resolution Data ◽  
Oligomeric Proteins ◽  
Oligomeric Protein ◽  
Accessible Surface ◽  
Chemical Cross Linking

Abstract Background The function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment. Results We present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function. Conclusions Our findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.

The Oct-1 POU domain mediates interactions between Oct-1 and other POU proteins

1992 ◽  
Vol 12 (2) ◽  
pp. 542-551
Author(s):  
C P Verrijzer ◽  
J A van Oosterhout ◽  
P C van der Vliet
Keyword(s):  
Dna Binding ◽  
Regulatory Proteins ◽  
Cross Linking ◽  
Dna Binding Domain ◽  
Specific Domain ◽  
Linker Region ◽  
Pou Domain ◽  
Protein Affinity ◽  
Gene Regulatory ◽  
Chemical Cross Linking

The POU domain is the conserved DNA binding domain of a family of gene regulatory proteins. It consists of a POU-specific domain and a POU homeodomain, connected by a variable linker region. Oct-1 is a ubiquitously expressed POU domain transcription factor. It binds to the canonical octamer sequence (ATGCAAAT) as a monomer. Here we show by chemical cross-linking and protein affinity chromatography that the Oct-1 POU domain monomers can interact in solution. This association requires both the POU homeodomain and the POU-specific domain. The interaction is transient in solution and can be stabilized by binding to the heptamer-octamer sequence in the immunoglobulin heavy-chain promoter. This correlates with cooperative DNA binding to this site. POU proteins from different subclasses, including Oct-1, Oct-2A, Oct-6, and a chimeric Oct-1 protein containing the Pit-1 POU domain, can bind cooperatively to a double binding site and form a heteromeric complex.

scholarly journals Stable Packaging of Phage PRD1 DNA Requires Adsorption Protein P2, Which Binds to the IncP Plasmid-Encoded Conjugative Transfer Complex

1999 ◽  
Vol 181 (21) ◽  
pp. 6689-6696 ◽  
Author(s):  
A. Marika Grahn ◽  
Javier Caldentey ◽  
Jaana K. H. Bamford ◽  
Dennis H. Bamford
Keyword(s):  
Filtration Rate ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
The Other ◽  
Cross Linking ◽  
Structural Protein ◽  
Phage Adsorption ◽  
Bacteriophage Prd1 ◽  
Double Stranded Dna ◽  
Chemical Cross Linking

ABSTRACT The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent Kd of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.

scholarly journals Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays

2019 ◽  
Author(s):  
Andrew Norris ◽  
Florian Busch ◽  
Michael Schupfner ◽  
Reinhard Sterner ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Early Time ◽  
Cross Linking ◽  
Tryptophan Synthase ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Primary And Secondary Metabolism ◽  
Chemical Cross Linking

The manuscript describes the use of chemical cross-linking/mass spectrometry and mutagenesis to investigate the dimeric interface of the tryptophan synthase α-subunit homolog, BX1. This work indicates that BX1 homodimerization might have served as a mechanism to exclude an interaction with the tryptophan synthase β-subunit, TrpB, at an early time in evolution, thereby eliminating cross-talk between primary and secondary metabolism. This work would be of interest to mass spectrometrists and structural biologist as it presents a workflow to determine the physiological protein-protein interactions within crystal structures using chemical cross-linking/mass spectrometry and mutagenesis as complementary structural biology techniques, thereby eliminating ambiguity and potential mis-assignments due to the presence of additional (artificial) protein contacts formed during the crystallization process.

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