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M T Fisher

Publications and source records attributed to M T Fisher.

At least 19 recordsLinked to original sources
PubMed2001

A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment.

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.

Amino Acid Sequence↗
PubMed2001

Complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor-1.

Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein.

Adenosine Triphosphate↗
PubMed2001

Structural changes in GroEL effected by binding a denatured protein substrate.

In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in non-native conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone, and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring.

Chaperonin 60↗
PubMed2000

Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of chaperonins and osmolytes.

A new method that uses a combination of bacterial GroE chaperonins and cellular osmolytes for in vitro protein folding is described. With this method, one can form stable chaperonin-protein folding intermediate complexes to prevent deleterious protein aggregation and, using these complexes, screen a large array of osmolyte solutions to rapidly identify the superior folding conditions. As a test substrate, we used GSDelta468, a truncation mutant of bacterial glutamine synthetase (GS) that cannot be refolded to significant yields in vitro with either chaperones or osmolytes alone. When our chaperonin/osmolyte method was employed to identify and optimize GSDelta468 refolding conditions, 67% of enzyme activity was recovered, comparable with refolding yields of wild type GS. This method can potentially be applied to the refolding of a broad spectrum of proteins.

Chaperonin 10↗
PubMed2000

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement.

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Bacterial Proteins↗
PubMed1998

Partitioning of rhodanese onto GroEL. Chaperonin binds a reversibly oxidized form derived from the native protein.

The mammalian mitochondrial enzyme, rhodanese, can form stable complexes with the Escherichia coli chaperonin GroEL if it is either refolded from 8 M urea in the presence of chaperonin or is simply added to the chaperonin as the folded conformer at 37 degreesC. In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a constant maximum value as the GroEL concentration increased. Over the same time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentration of urea accelerated the inactivation and partitioning of rhodanese onto GroEL. These results suggest that the GroEL chaperonin may facilitate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL-bound rhodanese can be completely recovered upon addition of GroES and ATP. The reactivation kinetics and commitment rates for GroEL-rhodanese complexes prepared from either unfolded or native rhodanese were identical. However, when rhodanese was allowed to inactivate spontaneously in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP. Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native rhodanese and GroEL were incubated under anaerobic conditions. Thus, our results strongly suggest that the inactive intermediate that partitions onto GroEL is the reversibly oxidized form of rhodanese.

Chaperonin 60↗
PubMed1998

Changing the nature of the initial chaperonin capture complex influences the substrate folding efficiency.

For the chaperonin substrates, rhodanese, malate dehydrogenase (MDH), and glutamine synthetase (GS), the folding efficiencies, and the lifetimes of folding intermediates were measured with either the nucleotide-free GroEL or the activated ATP.GroEL.GroES chaperonin complex. With both nucleotide-free and activated complex, the folding efficiency of rhodanese and MDH remained high over a large range of GroEL to substrate concentration ratios (up to 1:1). In contrast, the folding efficiency of GS began to decline at ratios lower than 8:1. At ratios where the refolding yields were initially the same, only a relatively small increase (1.6-fold) in misfolding kinetics of MDH was observed with either the nucleotide-free or activated chaperonin complex. For rhodanese, no change was detected with either chaperonin complex. In contrast, GS lost its ability to interact with the chaperonin system at an accelerated rate (8-fold increase) when the activated complex instead of the nucleotide-free complex was used to rescue the protein from misfolding. Our data demonstrate that the differences in the refolding yields are related to the intrinsic folding kinetics of the protein substrates. We suggest that the early kinetic events at the substrate level ultimately govern successful chaperonin-substrate interactions and play a crucial role in dictating polypeptide flux through the chaperonin system. Our results also indicate that an accurate assessment of the transient properties of folding intermediates that dictate the initial chaperonin-substrate interactions requires the use of the activated complex as the interacting chaperonin species.

Chaperonins↗
PubMed1998

GroE chaperonin-assisted folding and assembly of dodecameric glutamine synthetase.

The folding and assembly of Escherichia coli dodecameric glutamine synthetase is facilitated by the E. coli GroE chaperonins, GroEL and GroES. Since endogenous glutamine synthetase monomers are bound to GroEL immediately after cell lysis and are assembly competent, this strongly suggests that glutamine synthetase is an authentic substrate of the GroE chaperonins. At physiological temperatures, the in vitro reactivation of glutamine synthetase increases from 10 to 70-80% of the original activity when the chaperonin GroEL is included. Although nucleotide binding is sufficient to dissociate assembly competent glutamine synthetase monomers from GroEL, the addition of GroES substantially accelerates the dissociation, assembly, and reactivation. The interactions of glutamine synthetase monomers with the activated chaperonin are transient (t1/2 = 10 sec) and these monomers can be released from GroEL at high concentrations without misfolding or inappropriate aggregation. It has been found that the nucleotide-induced conformational change of GroEL is critical for folding success of glutamine synthetase because the simple displacement of glutamine synthetase monomers from the GroEL chaperonin with another protein substrate inhibits reactivation. During glutamine synthetase refolding, the "high affinity" nucleotide-free GroEL is most efficient in preventing initial folding intermediates from partitioning to off-pathway folding routes. Interestingly, the more physiologically relevant "low affinity" nucleotide-bound ((ATP/ADP) GroEL--GroES) complex is not as efficient at capturing the initial folding intermediates of glutamine synthetase. In contrast to glutamine synthetase, non-authentic "model" substrates such as mammalian mitochondrial rhodanese and mitochondrial malate dehydrogenase show no differences in folding efficiencies with either the "low affinity" or "high affinity" complexes. Besides the nature of the chaperonin complex itself, the mechanism of GroE-assisted folding is determined by the folding environment and, most importantly, by initial interactions of chaperonins with folding intermediates. Glutamine synthetase interacts only transiently with chaperonin complexes, while most of the "model" proteins exhibit relatively long interactions times. It may be indicative of a specific evolutionary selected mechanism of chaperonin-assisted folding (optimizing the folding kinetics), different from that observed with non-authentic chaperonin substrates. Since the kinetics of protein folding depends heavily on the solution environment, studies involving in vivo chaperonin substrates under conditions that closely mimic those found in the cell will be required to define and solve the physiologically relevant kinetic mechanism of chaperonin-assisted folding.

Bacterial Proteins↗
PubMed1995

Interactions between the GroE chaperonins and rhodanese. Multiple intermediates and release and rebinding.

Efficient renaturation of urea-denatured rhodanese using the chaperonin GroE system requires GroEL, GroES, and ATP. At high concentrations this renaturation also requires the substrate thiosulfate to have been present during GroEL-rhodanese complex formation. When thiosulfate is present the GroEL-rhodanese complex can be concentrated to greater than 1 mg/ml rhodanese with little effect on the efficiency of renaturation. However, if complex is formed in the absence of thiosulfate, renaturation of rhodanese in the presence of thiosulfate shows a critical concentration of approximately 0.4 mg/ml, above which renaturation yields drop dramatically. This critical concentration appears to be related to an aggregation event in the refolding of rhodanese. The nucleotide free or ADP-bound form of GroEL also binds to rhodanese that has been either already renatured or never denatured. The bound rhodanese has no activity but can be released from GroEL with ATP recovering 90% of control activity. The data presented herein support a release and rebinding mechanism for the GroE-assisted refolding of rhodanese. It also suggests GroEL binds several protein folding intermediates along the entire refolding pathway.

Adenosine Diphosphate↗
PubMed1994

The rates of commitment to renaturation of rhodanese and glutamine synthetase in the presence of the groE chaperonins.

Current models of chaperonin-assisted folding suggest that proteins undergo multiple rounds of binding and release before they are released in a form that is committed to folding to the native state. Using immunoprecipitation techniques, we have determined the rates at which rhodanese and glutamine synthetase (GS) are released from groEL in a form committed to refold to active enzyme. Rhodanese and glutamine synthetase were chosen as substrates because they exhibit different solution requirements for the chaperonin system and they form stable "folding arrested" complexes with groEL. At various times during the groE-dependent renaturations, groEL was rapidly removed from the renaturation mixture by immunoprecipitation and centrifugation (30 s). The conformers that are committed to the native state remained in the supernatant and were assayed after 1 h. At 25 degrees C, the rate profiles indicate the release and commitment to folding of GS to its native state occurs far earlier (t1/2 < 1 min) than for rhodanese (t1/2 = 5 min). In light of previous results, it appears that GS monomers can attain a groE-independent assembly competent conformation after a brief interaction with the chaperonin. In contrast, the renaturation rate for rhodanese with the groE chaperonins mirrored the committed renaturation rates following groEL depletion. This suggests that rhodanese must interact with groEL throughout most of its folding reaction before it acquires a folding competent (groE independent) state. If current models of chaperonin mechanism are correct, rhodanese undergoes more rebinding and release cycles than does GS. Structurally, the degree of cycling and hence the rate of commitment to folding to the active form are probably dictated by the hydrophobic nature, number, and lifetimes of the folding intermediates that interact with the chaperonins.

Chaperonin 60↗
PubMed1994

Refolding and release of tubulins by a functional immobilized groEL column.

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.

Adenosine Triphosphate↗
PubMed1994

The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP.

The yields of active dodecameric glutamine synthetase (GS) are significantly increased when in vitro folding is initiated in the presence of the Escherichia coli groE chaperonins and ATP (37 degrees C). To observe the effects of chaperonins and ATP on GS assembly, the GS assembly intermediates were separated by nondenaturing gel electrophoresis, visualized by Western analysis, and studied as a function of time. The form of GS that was initially released from groEL is monomeric. After the monomers formed dimers, active GS oligomers were assembled by the association of assembly competent dimers with higher order even-numbered oligomers until the dodecamer was formed. When ATP was added to the groEL.GS complex (no groES), a groEL.GS complex remained visible for up to 30 min after the renaturation was initiated. This slow disappearance of the groEL.GS complex is consistent with observed lags in both the GS activity regain profile and the assembly-dependent increase in GS tryptophan fluorescence. When groES was present, the addition of ATP resulted in the disappearance of observable complex at early sample times (< 2 min). Concomitantly, the rates of the regain of GS activity and the GS-dependent increase in tryptophan fluorescence intensity showed substantial accelerations. These results indicate that groES facilitates GS assembly from groEL by inducing the rapid release of GS from groEL, which in turn increases the concentration of assembly competent GS monomers. In addition, groES can initiate renaturation of GS from the groEL.GS arrested complex in the presence of ADP. When chaperonin-dependent GS renaturation was initiated with ATP or ADP (> or = 2 mM), the rates were identical. Since ATP hydrolysis is not absolutely required, the combined binding energies of groES and ATP (or ADP) appear to be sufficient to weaken the binding affinity of groEL for GS subunits and facilitate the release and refolding of assembly competent GS monomers from groEL.

Adenosine Diphosphate↗
PubMed1993

On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes.

For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions. Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T. (1992) Biochemistry 31, 3955-3963). In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins. When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline. The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent. It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate. If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly.

Bacterial Proteins↗
PubMed1992

Promotion of the in vitro renaturation of dodecameric glutamine synthetase from Escherichia coli in the presence of GroEL (chaperonin-60) and ATP.

The folding and assembly of dodecameric glutamine synthetase (GS) from Escherichia coli was examined in the absence and presence of the E. coli heat shock protein, GroEL (chaperonin-60). At nonphysiological temperatures (15-20 degrees C), unfolded GS spontaneously renatured to 80-90% of its original activity in the absence of GroEL. At near-physiological temperatures (37 degrees C), only 20-40% of the original activity returns. Under the latter solution conditions, GroEL and ATP enhance the extent of GS renaturation to 70-80% of the original activity at 37 degrees C. In the absence of ATP, GroEL arrests the renaturation of unfolded GS by forming a stable binary complex. The addition of ATP to this complex resulted in the release of GS subunits and formation of active dodecameric GS. The order of addition of ATP or unfolded GS to GroEL results in differences in the t1/2 values where half-maximal GS activity is attained. At a constant GS concentration, the formation of the GroEL.GS complex followed by ATP addition resulted in approximately a 2-fold increase in the observed t1/2 value compared to that observed when GroEL was preincubated with ATP before the GS renaturation reaction was initiated. These differences in renaturation rates may be related to binding affinity differences between the ATP-free and -bound GroEL conformer for unfolded or partially folded protein substrates [Badcoe, I. G., Smith, C. J., Wood, S., Halsall, D. J., Holbrook, J. J., Lund, P., & Clarke, A. R. (1991) Biochemistry 30, 9195-9200].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.

Adenosine Triphosphate↗
PubMed1991

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli.

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.

Cytochrome b Group↗
PubMed1990

Xenopus transcription factor IIIA. Evidence for heterogeneity of Zn2+ binding affinities and specific labeling of cysteine 287.

The release of Zn2+ from transcription factor IIIA (TFIIIA) was examined with the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) in the absence and presence of p-hydroxymercuriphenylsulfonate (PMPS). With 0.5 mM PAR, approximately 5 eq of Zn2+ were released from TFIIIA, but no Zn2+ release was detected from the 7 S ribonucleoprotein. The PMPS-promoted Zn2+ release from TFIIIA was 8.7 +/- 0.4 eq of Zn2+ of which approximately 4 eq of Zn2+ rebound to TFIIIA upon displacement of the mercurial with excess 2-mercaptoethanol. These results suggest that at least two affinity classes of Zn2+ binding sites exist in TFIIIA, one of which is released to 0.5 mM PAR in the absence of PMPS. Also, 18 of the 23 cysteine residues of TFIIIA reacted with 5,5'-dithiobis-(2-nitrobenzoic acid). The kinetic data of PAR and 5,5'-dithiobis-(2-nitrobenzoic acid) reactions with TFIIIA were similar, and the spectral changes were characterized by at least three exponential terms. Both TFIIIA and the 7 S particle were reacted with the thiol-specific fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS). Complete trypsin hydrolysis followed by reverse-phase high pressure liquid chromatography analysis of peptide mixtures showed only one fluorescent peak from the AEDANS-labeled 7 S particle whereas numerous fluorescent peaks were observed with AEDANS-labeled TFIIIA. This further indicates exposure of cysteine residues from Zn2+ binding domains in TFIIIA. Cys287 was identified as the site of modification by amino acid sequencing of the isolated fluorescent peptide from the derivatized 7 S particle. Limited papain cleavage of the AEDANS-labeled 7 S particle indicated that the modified cysteine is located within a 34-kDa TFIIIA fragment. Gel retardation and transcription assays showed that TFIIIA, which had been purified from the AEDANS-labeled 7 S particle, was capable of binding to the internal control region of 5 S RNA gene and retained transcription activity. Thus, Zn2+ binding domains and all but 1 cysteine residue are buried in the 7 S particle, thereby facilitating site-specific labeling of TFIIIA.

Animals↗