Thermal Characterization and Interaction of the Subunits from the Multimeric Bacteriophage Endolysin PlyC.

Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small-molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating its long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic, and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46 °C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall-binding domain) is thermostable, denaturing at ~75 °C. The isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized, whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74 °C in phosphate-buffered saline with each subunit subsequently denaturing at 92 °C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

The role of Cys-298 in aldose reductase function.

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein.

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between beta7 and beta8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.

ATP hydrolysis and DNA binding confer thermostability on the MCM helicase.

The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 degrees C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 degrees C higher in the presence of DNA and either ATP or ADP-AlF(4)(-), a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPgammaS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA binding domain and the C-terminal catalytic domain in the presence of DNA.

Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase.

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 degrees C to 35 degrees C. At 25 degrees C and with an increase in the Pi concentration from 0 to 50mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 degrees C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol(-1) with increase in Pi concentration from 50 to 100mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 microM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 x 10(5) M(-1) to 1.14 x 10(5) M(-1). This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.

Effect of the distal C162S mutation on the energetics of drug binding to p38alpha MAP kinase.

The binding reactions of the inhibitor drugs, SB 203580, SKF 86002, and p38 INH.1 to the isoforms 1 and 2 splice variants of p38alpha MAP kinase and their C162S mutants, as determined from ITC measurements from 25 to 35 degrees C, are totally enthalpically driven with binding constants ranging from 10(7)M(-1) for SKF 86002 and SB 203580 to 10(9)M(-1) for p38 INH.1. Interactions of p38 INH.1 with an additional hydrophobic pocket of the kinase would account for its large increase in K(b). DSC scans exhibited single unfolding transitions for the isoforms, their mutants, and the mutants bound to the drug inhibitors. Two transitions, however, were observed for the isoform-drug complexes of SB 203580 and p38 INH.1 and were attributed to decoupled unfolding of the N- and C-terminal domains of the kinase. The C-terminal domain of isoform 1 is estimated to be less stable than of isoform 2 by 15 kJ mol(-1).

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength.

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.

High-resolution crystal structures of Erythrina cristagalli lectin in complex with lactose and 2'-alpha-L-fucosyllactose and correlation with thermodynamic binding data.

The primary sequence of Erythrina cristagalli lectin (ECL) was mapped by mass spectrometry, and the crystal structures of the lectin in complex with lactose and 2'-alpha-L-fucosyllactose were determined at 1.6A and 1.7A resolution, respectively. The two complexes were compared with the crystal structure of the closely related Erythrina corallodendron lectin (ECorL) in complex with lactose, with the crystal structure of the Ulex europaeus lectin II in complex with 2'-alpha-L-fucosyllactose, and with two modeled complexes of ECorL with 2'-alpha-L-fucosyl-N-acetyllactosamine. The molecular models are very similar to the crystal structure of ECL in complex with 2'-alpha-L-fucosyllactose with respect to the overall mode of binding, with the L-fucose fitting snugly into the cavity surrounded by Tyr106, Tyr108, Trp135 and Pro134 adjoining the primary combining site of the lectin. Marked differences were however noted between the models and the experimental structure in the network of hydrogen bonds and hydrophobic interactions holding the L-fucose in the combining site of the lectin, pointing to limitations of the modeling approach. In addition to the structural characterization of the ECL complexes, an effort was undertaken to correlate the structural data with thermodynamic data obtained from microcalorimetry, revealing the importance of the water network in the lectin combining site for carbohydrate binding.

Energetic contributions to the initiation of transcription in E. coli.

The thermodynamics of RNA polymerase (RNAP) binding to a 108 base pair (bp) synthetic promoter with consensus sequences at the -35 and -10 bp binding regions upstream from the transcription start point were determined using isothermal titration calorimetry (ITC). The binding constant at 25 degrees C is 2.37+/-0.18x10(7) M(-1), which is reduced to 0.17+/-0.06x10(7) M(-1) with mutations in the -10 bp region but remained the same with mutations in the -35 binding region. The binding reactions were enthalpically-driven with exothermic binding enthalpies ranging from -57+/-6 kJ mol(-1) at 15 degrees C to -271+/-20 kJ mol(-1) at 35 degrees C yielding a large binding heat capacity change of -10.7+/-1.9 kJ mol(-1) K(-1), indicating a conformational change upon binding to the RNAP. Differential scanning calorimetry (DSC) scans of the thermal unfolding of RNAP and the promoter-RNAP complex exhibited an unfolding transition at 55.5+/-0.6 degrees C and at 58.9+/-0.5 degrees C for the RNAP but only one transition at 60.5+/-1.1 degrees C for the complex with van't Hoff enthalpy to transition enthalpy ratios of, resp., 3.2+/-0.3 and 4.3+/-0.5. The single transition of the complex results from a shift to 60.5 degrees C of the low temperature transition upon promoter binding to the structural unit unfolding at the lower temperature in RNAP. The large transition enthalpy ratios indicate that the sigma, alpha, alpha, beta, and beta' subunits unfold as almost independent entities. The dissociation thermodynamics of short transcription "bubble" duplexes of 7 promoters sequenced from -1 to -12 bp were determined from ITC and DSC measurements. The free energy change of the promoter binding to the RNAP and the free energy requirement for formation of the transcription bubble at the low promoter concentrations in the cell are sufficient to drive the initiation of transcription through the isomerization of the closed to the open form step of the RNAP-promoter complex.

Thermodynamics of single strand DNA base stacking.

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<=>U transition in this temperature range. The transition properties of the ssDNA sequences with

Thermal stabilization of human albumin by medium- and short-chain n-alkyl fatty acid anions.

A comprehensive study of the thermal stabilization of defatted human albumin monomer by n-alkyl fatty acid anions (FAAs), formate through n-decanoate, was carried out by differential scanning calorimetry (DSC). The concentration of each ligand affording maximum thermal stabilization was determined; n-nonanoate provides the greatest stabilization but is only marginally better than n-octanoate and n-decanoate. The use of reversible thermodynamics and a two-state denaturation model for albumin has been validated. Standard free energies of binding, calculated from increases in free energy of denaturation, for n-butanoate and longer FAAs, are linear with n-alkyl chain length whereas those for formate, acetate, and n-propionate deviate from linearity; those for acetate and n-propionate are even greater than that of n-butanoate, thereby suggesting, in addition to the common class of sites available to all such ligands, the presence of an additional class of lower affinity binding sites available only to these shortest ligands. Competition experiments involving acetate and n-octanoate and involving n-pentanoate and n-octanoate confirmed the binding of acetate to lower affinity sites unavailable to n-octanoate and n-pentanoate. Furthermore, an equation is provided, allowing computation of the transition temperature as a function of the free energy for any reversible process causing a change in thermal stability of a protein undergoing reversible, two-state denaturation. With this equation, modeling the competition experiments by using the binding parameters determined by DSC provides additional support for the class of lower affinity sites, which play a significant role in thermal stabilization of albumin at higher concentrations of these shortest FAAs.

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein.

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.

Importance of product/reactant equilibration in the kinetics of the phosphoglucose isomerization reaction by differential stopped flow microcalorimetry.

The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P --> G6P) by baker's yeast phosphoglucose isomerase (PGI) with regard to k(cat) and K(m) were determined from analysis of differential stopped flow microcalorimeter measurements using the integrated form of the Michaelis-Menten rate equation. Values for K(m) (F6P --> G6P) that were determined at pH 8.0 and ionic strength 0.1M at 293.4, 298.4, 303.4, and 311.5K exhibited a linear dependence on the substrate concentration at each temperature because of the substrate-product equilibrium. The minimum values for K(m) ranged from 2.62+/-0.55 mM at 293.4K to 7.8+/-4.8mM at 311.5K and were the same as the minimum values for the reverse reaction (G6P --> F6P) at 293.4 K and 298.4 K. Minimum values for k(cat) increased with temperature, from 2.78+/-0.34s(-1) at 293.4K to 11.4+/-1.0s(-1) at 311.5K, and for the reverse reaction, G6P --> F6P, from 0.852+/-0.086 s(-1) at 293.4K to 1.46+/-0.06s(-1) at 298.4K. The enzyme efficiency at 311.5K is close to the collision rate for a diffusion-controlled process in solution. The [F6P]/[G6P] equilibrium constants were determined from comparison of the values of k(cat) in both directions and were 0.307+/-0.053 at 293.4K and 0.395+/-0.033 at 298.4K. The heats of reaction in the F6P --> G6P direction increased from -8.96+/-0.26 kJmol(-1) at 311.5K to -8.27+/-0.40 kJmol(-1) at 293.4K, a value in fair agreement with 7.01+/-0.32 kJmol(-1) in the opposite G6P --> F6P direction.

Temperature dependence of the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.

The rates of the hydrolysis of N-acetylglycine, N-acetyl-L-methionine, and N-acetyl-L-phenylalanine by porcine acylase I in 0.1M phosphate buffer, which is inhibited by acetate product formation, were monitored calorimetrically at temperatures between 15.2 and 45.3 degrees C by a differential stopped-flow microcalorimeter. Since the acylase is thermally stable and the pH of the phosphate buffer changes by less than 0.01 pH units over this temperature range, any temperature effect on the hydrolysis reaction can be attributed to the effect of temperature on the kinetics of the reaction. Analysis of the integrated heat released by the reaction as a function of time with regard to the integrated Michaelis-Menten equation yields apparent values for k(cat) and apparent values for Km that depend on the product inhibition constant. The apparent values for Km also exhibited a dependence on the initial substrate concentration because of the acetate product inhibition at each temperature. By assuming that the inhibition constant is independent of temperature over this temperature range and from extrapolation of Km to its value at zero substrate concentration, intrinsic values of Km and k(cat) were determined over the temperature range from 15.2 to 45.3 degrees C. The intrinsic values of Km exhibited very little variation over this temperature range while the intrinsic values of k(cat) exhibited an increase over the same temperature range. The heats of reaction also exhibited an increase with temperature over this range with an average heat capacity change of -94 Jmol(-1)K(-1) for the three substrates.

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.

Entropic nature of the interaction between promoter bound CRP mutants and RNA polymerase.

The interaction between CRP, T127L, S128A, and CRP and RNA polymerase bound to a 104 bp synthetic promoter were determined by ITC at 298 K and ranges from a deltaG(b) degrees = 1.4 +/- 0.8 kJ mol(-)(1) (cAMP-ligated S128A) to 4.5 +/- 0.3 kJ mol(-)(1) (cAMP-ligated double mutant CRP) with endothermicities that range from 4 +/- 3 kJ mol(-)(1) (cAMP-ligated CRP) to 47 +/- 8 kJ mol(-)(1) (cGMP-ligated T127L). The interaction is, thus, entropically driven, exhibits enthalpy-entropy compensation, and increases the binding affinity of the RNA polymerase to the promoter by factors ranging from 1.7 +/- 0.1 (cAMP-ligated S128A) to 6.1 +/- 0.1 (cAMP-ligated CRP). Although the binding affinities to the promoter alone, except for cAMP-ligated S128A, are the same as to a shorter 40 bp duplex containing the same CRP consensus binding site sequence (conDNA), the binding enthalpies of CRP/mutant to the promoter are lower by factors of 2-3 x than the corresponding binding enthalpies to conDNA. Small angle neutron scattering measurements on the DNA-CRP/mutant complexes in D(2)O/H(2)O solutions exhibit an increase in the Rg of the CRP/mutant component from 22 to 27-31 A that can be attributed to a conformational change in the N-terminal domain of CRP. The Rg = 27 A for the bound conDNA can be attributed to a slight unwinding of the DNA in solution that would also enhance the activation of transcription. The Rg = 53 +/- 3 A for the bound promoter is attributed to bending of the promoter in solution that can be responsible for the lower CRP/mutant-promoter binding endothermicities.

Effects of metal ligation and oxygen on the reversibility of the thermal denaturation of Pseudomonas aeruginosa azurin.

Thermodynamic equilibrium transition models in DSC are only applicable to reversible processes, but reversibility of the thermal transitions of proteins is comparatively rare because of intermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures. The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversible thermal denaturation, both as holo- and apoprotein [Engeseth, H. R., and McMillin, D. R. (1986) Biochemistry 25, 2448-2455]. In this study, however, we demonstrate that this beta-barrel protein of Greek key topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligated to the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusively oxidized in a transition metal catalyzed oxidation reaction with dissolved O(2) at high temperatures. Both Cu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as well as the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant. Correspondingly, apoazurin mutants C112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-state transitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due to autoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.

Design and use of a peptide nucleic acid for detection of the heteroplasmic low-frequency mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) mutation in human mitochondrial DNA.

BACKGROUND: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. METHODS: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. RESULTS: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence. CONCLUSION: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.