Segmental flexibility in an antibody molecule

Yguerabide, Juan; Epstein, Henry F.; Stryer, Lubert

doi:10.1016/0022-2836(70)90009-4

Cited by 470 publications

(228 citation statements)

References 37 publications

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“…This indicates that the binding of two rather heavy antigen molecules partially restricts segmental flexibility as observed by Yguerabide et al [24] in the case of hapten labelled antibodies.…”

Section: Resultssupporting

confidence: 63%

X‐ray small‐angle scattering on soluble antigen—antibody complexes

et al. 1971

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Section: Resultssupporting

confidence: 63%

X‐ray small‐angle scattering on soluble antigen—antibody complexes

et al. 1971

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“…Hence, the global rotation reorientation time obtained in this reaction predominantly represents that of the protein-DNA complex. Using the rotation reorientation time Global ϭ 48.03 Ϯ 10.05 ns obtained under saturation binding conditions and the original Perrin equation (Equation 7; see "Experimental Procedures"), we can estimate the size of the StrRAG1⅐12-RSS complex to be between Ϸ175 kDa (using h ϭ 0 ml/g, an anhydrous sphere) and 120 kDa (using h ϭ 0.4 ml/g, the maximal degree of hydration) (46,47). Given the apparent molecular masses of the protein (72 kDa) and 12-RSS-Fl DNA (31 kDa), we conclude that StrRAG1 is most likely present as a dimer in these complexes.…”

Section: Resultsmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

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“…The Fab arms flexibility was shown several years ago by steady-state fluorescence polarization measurements of DNS-immunoglobulins, their fragments and specific complexes of anti-DNS antibody with DNShapten [6,7,1 l] as well as by the ns technique [12]. Recently these observations were confirmed by ns fluorescent spectroscopy measurements of specific antibody complexes with another fluorescence dye, pyrenylbutyrate [ 131.…”

Section: Comparison Of Ph For Precipitating and Nonprecipitating Pig mentioning

confidence: 95%