Comparison of the dimensions of the combining sites of the dinitrophenyl-binding immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 by spin-label mapping

Willan, Keith J.; Marsh, Derek; Sunderland, C.A.; Sutton, Brian J.; Wain‐Hobson, Simon; Dwek, Raymond A.

doi:10.1042/bj1650199

Cited by 18 publications

(5 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Consequently, for haptens (V) and (VI), only the larger of the two A_,,, values in each case will require allowance for the effect of slow molecular tumbling. This interpretation is supported by a study of hapten binding to another myeloma protein (MOPC 460) and its fragments, in which the site applies no constraints on the hapten's mobility, and A I z is independent of the fragment's size (Willan et al, 1977 Table 4. Maximum hyperfine splittings, A.,., (mT)ofspin-labelledhaptens bound tofragments ofmyelomaprotein MOPC 315…”

Section: Results From Different Fragmentsmentioning

confidence: 88%

“…In this study there are no marked discontinuities in the e.s.r. spectra between the different haptens, and the binding constants (Willan et al, 1977) show no evidence for such specific interactions.…”

Section: Methods Of Calculation Haptenmentioning

confidence: 91%

“…The results of this analysis, the Euler angles relating these motions to the nitroxide radical itself, the calculations of the principal values of the averaged A tensor and the spatial requirements for the motions, are presented in Tables 1 and 2. These Tables are of general applicability for any Dnp-binding protein (Willan et al, 1977).…”

mentioning

confidence: 99%

See 2 more Smart Citations

The gross architecture of an antibody-combining site as determined by spin-label mapping

Sutton

Gettins

Marsh

et al. 1977

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ‘tumbling’ time of the antibody–hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1–1.2nm and has lateral dimensions at the entrance to the site ≥0.6nm×0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95L (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody–hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.

show abstract

Section: Results From Different Fragmentsmentioning

confidence: 88%

Section: Methods Of Calculation Haptenmentioning

confidence: 91%

See 1 more Smart Citation

The gross architecture of an antibody-combining site as determined by spin-label mapping

Sutton

Gettins

Marsh

et al. 1977

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…This value, which is only an approximation, was obtained by adding the maximal distance of the DNP antigen from the 3' hydroxyl of tRNA (about 15 A) to the distance of the 3' end from the anticodon, 76-83 A (25), and subtracting 12 A, which is the distance of the DNP binding site from the periphery of the Ab (26). This relatively long distance may explain why apparent multiple binding sites were found from a supposed single point of attachment.…”

Section: Resultsmentioning

confidence: 99%

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

Keren-Zur¹,

Boublík²,

Ofengand³

1979

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA al, derivatized at the a-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310-to 325-nm irradiation. Previous work has shown that the crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. We recently observed that Eschertchia coli AcVal-tRNAlal (and certain other tRNAs) bound at the P site of 70S ribosomes could be covalently crosslinked with high efficiency to 16S ribosomal RNA by irradiation at 310-325 nm (13). This reaction linked the anticodon loop of the tRNA to the 3' one-third of the 16S ribosomal RNA (15,16). Since the tRNA was covalently attached via its anticodon to the presumptive decoding region of the ribosome, the possibility of visualization of this site by EM was considered. Preliminary experiments showed that it was not possible to directly visualize tRNA on the ribosome with the present EM methods. We decided, therefore, to attach a suitable antigenic determinant to the tRNA in order to use antibody (Ab) against it as a marker for the location of the tRNA molecule. This technique, which we call affinity immuno-EM, has provided a direct approach to localization of the decoding region on the 30S particle. MATERIALS AND METHODSN-(y-Dinitrophenyl)aminobutyryl (DNP-AbuX3HJVal-tRNA. DNP-Abu (Sigma) was coupled to N-hydroxysuccinimide (HONSu) with N,N'-dicyclohexylcarbodiimide in dimethylformamide. Esterification was followed by silica gel thin-layer chromatography (Eastman) developed with ethyl acetate/methanol (10:1). The RF of DNP-Abu = 0.1; RF Of DNP-Abu-ONSu = 0.9. After isolation, the product was tested by reaction with glucosamine. There was complete conversion to a new product, possessing an RF of 0.48 (the RF of DNP-Abu was 0.86 and the RF of DNP-Abu-ONSu was 0.9) when analyzed by silica gel thin-layer chromatography developed with chloroform/methanol/HOAc (65:30:5 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

show abstract

“…Proteins M460 and X25 have Kd values of 3.3 gM (Jaffe et al, 1971) and 5,M (Sharon & Givol, 1976) respectively, and might have been expected to behave similarly on the basis of affinity but differently from protein M315, with its Kd of 0.1 pM. Willan et al (1977), in comparing the dimensions of the combining sites of these three proteins by e.s.r. studies on nitroxide spin-labelled Dnp haptens, found that the entrance to the Dnp subsite, presumably the volume occupied by the lysine side chain, was much wider in protein M460 than in either protein X25 or protein M315.…”

Section: Dnp-glymentioning

confidence: 99%

The variability of nitro group-protein interaction in the 2,4-dinitrophenyl-binding antibodies M315, M460 and X25 investigated by resonance Raman spectroscopy

Gettins¹,

Dwek²,

Perutz³

1981

Biochemical Journal

View full text Add to dashboard Cite

Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab' and X25 Fab'. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N--C--C--2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab' and X25 Fab' fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline.

show abstract

Comparison of the dimensions of the combining sites of the dinitrophenyl-binding immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 by spin-label mapping

Cited by 18 publications

References 13 publications

The gross architecture of an antibody-combining site as determined by spin-label mapping

The gross architecture of an antibody-combining site as determined by spin-label mapping

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The variability of nitro group-protein interaction in the 2,4-dinitrophenyl-binding antibodies M315, M460 and X25 investigated by resonance Raman spectroscopy

Contact Info

Product

Resources

About