György Hegyi¹, Marianna Mák², Eldar Kim³, Marshall Elzinga⁴, Elena Bobkova³, Carl J. Miller³, Albina Orlova⁵, Edward H. Egelman⁵, Andras Muhlrad³, and Emil Reisler³

1. Department of Biochemistry, Eötvös Loránd University, Budapest Hungary,
2. Spectroscopic Research Division, Gedeon Richter Ltd Budapest Hungary,
3. Department of Chemistry and Biochemistry UCLA Los Angeles ,
4. New York State Institute for Basic Research in Developmental Disabilities, New York,
5. Departmant of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis

Chemical cross-linking can give information about the proximity’s of particular sites on proteins or protein assemblies and about the functional consequences of the new covalent bounds introduced by such a reaction. In the experiments reported here, two heterobifunctional photo activable cross-linking reagents were designed, synthesised, and covalently bound to rabbit skeletal muscle actin. Both reagent have a photoactivable arylazido group on one side and a primary amine on the other side. The amine group can be attached to an exposed glutamine side chain by a transglutaminase (TGase) catalysed reaction, then the arylazido moiety can be photoactivated to make the second step in the cross-linking reaction. However the two reagents have different length, orientation and photochemical parameters.

The first reagent 4-azidobenzoyl-putrescine (ABP) was incorporated to G actin up to 0.5 M/M, whereas F actin was refractory to ABP incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labelled G actin was polymerised, and irradiation of the F actin led to a covalent intermolecular cross-linking of the adjacent monomers in the actin filaments. Peptide mapping and sequence analysis, as well as mass spectroscopy, indicate that the actual cross-linking was between Gln-41 and Lys 113. Thus the g -carboxyl group of Gln-41 must be within 10.7 Å from the Lys 113 in the adjacent actin monomer, which is in good agreement with the atomic model for F actin proposed by Holmes et al. The substochiometric incorporation of the reagent and the relative low yield of the photogenerated cross-linking did not allow the study of the functional consequences of this reaction. Therefore, an improved reagent, 4-azido 2-nitrophenyl-putrescine (ANP), was synthesised for the second series of experiments.

The new reagent has better photochemical properties, and using a bacterial TGase instead of guinea pig liver TGase, 1 M/M incorporation and improved yield of cross-linking were found. Peptide analysis of the cross-linked actin indicated that the cross-linking took place between Gln 41 and Cys 374 . The dynamic length of ANP, between 11.1 and 12.5Å, constrains to that range the distance between the g -carboxyl group of Gln-41 in one monomer and the sulphur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in F-actin predicted by the atomic models of Holmes et al. and Lorenz et al.

Structural and functional properties of intrastrand cross-linked actin filaments (between Gln-41 and Cys-374) were also examined. Extensively cross-linked F-actin (88% xross-linked) was inactivated at 60^o C at a ten fold slower rate than the uncross-linked actin. Similarly, in CD melting experiments the unfolding of the cross-linked actin took place at a temperature higher by 11^o C from that observed for control actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure, however a slightly different orientation of subdomain 2 was observed. Rigor and weak myosin subfragment (S1) binding and acto-S1 ATPase measured with 50 and 90% ANP cross-linked F-actin did not show major changes in these functions compared to the uncross-linked actin; the K_d and K_m values were little affected by the cross-linking, and the V_max decreased only by 50% for the extensively cross-linked actin. The cross-linking of actin, however, strongly inhibited the generation of motion and force by heavy meromyosin (HMM) in the in vitro motility assays. The mean speed of actin filaments decreased with the increase in their cross-linking and approached zero for 90% cross-linked actin.

Actin filaments partially cross-linked with ANP can be depolymerized and fractionated into pools of longitudinal cross-linked dimers, trimers, and higher order oligomers. The cross-linked dimers, trimers, and oligomers were polymerized i.e. reassembled into filaments by MgCl₂ faster than uncross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than the uncross-linked actin filaments. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behaviour in the in vitro motility assays. Filaments made from cross-linked dimers moved at 50% speed of the uncross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely and the oligomers-made filaments did not move at all. These results show an uncoupling between force generation and other events in actomyosin interaction and emphasise the role of actin filament dynamics in the contractile process.