Hydrophobic loop dynamics and actin filament stability

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.     

Conformational dynamics of loop 262-274 in G- and F-actin

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.     

Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F- actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin''s ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.     

Antagonistic effects of cofilin, beryllium fluoride complex, and phalloidin on subdomain 2 and nucleotide-binding cleft in F-actin

Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound epsilon-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of epsilon-ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2.     

Intermolecular dynamics and function in actin filaments

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.     

Drebrin-induced Stabilization of Actin Filaments

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G)

Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin. In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Cofilin (ADF) affects lateral contacts in F-actin

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.     

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchanger

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes.

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Structural basis for CD44 recognition by ERM proteins

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.     

Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: effective design of a hybridization-sensitive probe

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.     

Fluorescent probe studies of normal, persistently infected, rous sarcoma virus-transformed, and trypsinized rat cells

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine were used to study membranes of normal cells, RSV-transformed cells, cells treated with a proteolytic enzyme, and cells persistently infected with lymphocytic choriomeningitis virus. The lifetimes of excited pyrene and pyrene butyric acid showed only minor changes when these probes were in normal, transformed, trypsinized or persistently infected cells. However, pyrene, but not pyrene butyric acid, lifetimes are shorter in cell membranes than in homogeneous solvents. The quenching of excited pyrene in cells by quencher molecules was slower than corresponding reactions in homogeneous solutions indicating that the probe was screened from the quenchers by the membrane. However, quenching reactions with the pyrene butyric acid probe were similar in cells and homogeneous solvents. This indicates that pyrene and pyrene butyric acid reside in different lipid regions of the membrane. Transformed and trypsinized cells showed increased membrane fluidity compared to normal and persistently infected cells. Membrane fluidity was determined from the excimer/monomer fluorescence ratios of pyrene, and by the polarization of N-phenyl 1-naphthylamine fluorescence. Several techniques distinguished between normal and transformed or trypsinized cells; however, the only parameter unique to viral transformation was a blue shift of the fluorescence maxima of N-phenyl 1-naphthylamine. This shift reflected a less polar environment for N-phenyl 1-naphthylamine in virus-transformed cells.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes

The fluorescent probes pyrene, pyrene butyric acid and $\text{N-}\text{phenyl}$ 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe $\text{N-}\text{phenyl}$ 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrne probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

The effect of overhanging nucleotides on fluorescence properties of hybridising oligonucleotides labelled with Alexa-488 and FAM fluorophores

In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3' overhang (4 bases), was hybridised to either 5' fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

A fluorescence-based detergent binding assay for protein hydrophobicity

Protein hydrophobicity is often detected by binding of protein to micelles of a mild detergent. A new fluorescence method for detection of this binding is presented. The method is based on a long-range quenching of tryptophan fluorescence by energy transfer to a pyrene-labeled phospholipid probe incorporated into micelles of Brij 96. The method is rapid, simple, and requires only a few micrograms of protein. Strongest quenching is obtained when both pyrene probe and brominated Brij 96, a short-range quencher, are combined. To define the best assay conditions the physical properties and quenching behavior of micelles with or without these probes have been compared. It is shown that both quenchers accurately measure binding of model compounds and protein toxins to micelles. Comparison of quenching by the different probes can be used to derive information on tryptophan location relative to the micelle core.     

F-Actin Structure Destabilization and DNase I Binding Loop Fluctuations: Mutational Cross-Linking and Electron Microscopy Analysis of Loop States and Effects on F-Actin

The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues > 45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (> 41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.