The Polyelectrolyte Behavior of Actin Filaments: A 25 Mg NMR Study ² Wujing Xian,* ,‡,§ Jay X. Tang, | Paul A. Janmey, | and William H. Braunlin ‡,⊥ Department of Chemistry, The UniVersity of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, and Hematology DiVision, Brigham and Women’s Hospital, 221 Longwood AVenue, Boston, Massachusetts 02115 ReceiVed September 24, 1998; ReVised Manuscript ReceiVed March 26, 1999 ABSTRACT: Under physiological conditions, filamentous actin (F-actin) is a polyanionic protein filament. Key features of the behavior of F-actin are shared with other well-characterized polyelectrolytes, in particular, duplex DNA. For example, the bundle formation of F-actin by polyvalent cations, including divalent metal ions such as Mg 2+ , has been proposed to be a natural consequence of the polyelectrolyte nature of actin filaments [Tang and Janmey (1996) J. Biol. Chem. 271, 8556-8563]. This recently proposed model also suggests that weak interactions between F-actin and Mg 2+ ions reflect a nonspecific trapping of counterions in the electric field surrounding F-actin due to its polyelectrolyte nature. To test this hypothesis, we have performed 25 Mg NMR measurements in F-actin solutions. Based on the NMR data, we estimate that the rotational correlation times of Mg 2+ are independent of the overall rotational dynamics of the actin filaments. Moreover, competitive binding experiments demonstrate a facile displacement of F-actin-bound Mg 2+ by Co(NH 3 ) 6 3+ . At higher Co(NH 3 ) 6 3+ concentrations, a fraction of the magnesium ions are trapped as actin filaments aggregate. ATP also competes effectively with actin filaments for binding to Mg 2+ . These results support the hypothesis that magnesium ions bind loosely and nonspecifically to actin filaments, and thus show a behavior typical of counterions in polyelectrolyte solutions. The observed features mimic to some extent the well-documented behavior of counterions in DNA solutions. Many biological macromolecules are polyelectrolytes, for example, DNA, RNA, charged polysaccharides, filamentous protein assemblies such as F-actin and microtubules, and viruses such as the bacteriophage fd and the tobacco mosaic virus (TMV). In solution, counterions accumulate in the vicinity of polyelectrolytes to balance the local charge. Several useful polyelectrolyte theories have been developed based on the cylindrical-rod cell model, which in its primitive form postulates that electrical charges are distributed uni- formly along the length of the polyelectrolyte (1). A specific polyelectrolyte is characterized by a dimensionless linear charge density, , defined as the ratio between the Bjerrum length λ B and the linear charge spacing b on the polyelec- trolyte. In the classical theory of simple electrolyte solutions, the Bjerrum length is a characteristic interaction distance for ion-pair formation (2), defined by where e is the elementary charge, kT is the thermal energy, ǫ 0 is the permittivity of vacuum, and ǫ is the relative dielectric constant. λ B is 7.1 Å in water at 20 °C with a dielectric constant ǫ ) 80. The counterion condensation (CC) theory of Manning provides a very useful quantitative description of the key features of polyelectrolyte-counterion interactions (3-6). The thermodynamic predictions of the Manning theory and the Poisson-Boltzmann theory (7, 8) are identical in the limit of infinite dilution. These two theories and other approaches (1, 9, 10) differ in the details of counterion distribution, but are in qualitative agreement on the existence of steep counterion gradients surrounding the polyelectrolyte. Since the key features of our argument are independent of such details, we will discuss our results in terms of the conceptu- ally attractive framework provided by CC theory. According to CC theory, there exists a critical charge density  crit ) 1, above which counterions condense in a thin layer surrounding the cylinder to maintain this critical value. Such a population represents a well-defined fraction (1 - 1/Z) of the total polyelectrolyte charge, where Z is the valence of the counterion. For example, for duplex DNA, b ) 1.7 Å; thus,  ) 4.2, and the total phosphate charge is neutralized to an estimated 77% by the monovalent cations, or 88% if sufficient divalent cations are present in solution. Filamentous (F)-actin is comprised of actin monomers of molecular mass 42 000 daltons, bound by specific nonco- valent self-assembling sites to form a double-helical filament. Each subunit of an actin filament contains one high-affinity (K d in the nanomolar range) divalent cation binding site that is usually occupied by Mg 2+ in vivo. Saturation of this binding site is insufficient to promote actin polymerization, which is driven either by millimolar concentrations of Mg 2+ or else by the order of 100 mM concentrations of monovalent ions. Assuming the amino acid sequence of R-skeletal muscle actin, each monomer subunit retains roughly 11 excess ² This work was supported by NIH grants to P.A.J. (AR38910) and W.H.B. (GM40438), and by an NIH training grant to J.X.T. (HL19429). * Corresponding author. ‡ The University of Nebraska-Lincoln. § Current address: Hematology Division, Brigham and Women’s Hospital, LMRC 301, 221 Longwood Ave., Boston, MA 02115. | Brigham and Women’s Hospital. ⊥ Current address: GelTex Pharmaceuticals, Nine-Fourth Ave., Waltham, MA 02154. λ B ) e 2 4πǫ 0 ǫkT 7219 Biochemistry 1999, 38, 7219-7226 10.1021/bi982301f CCC: $18.00 © 1999 American Chemical Society Published on Web 05/12/1999