Monoclonal antibody-directed radioimmunoassay detects cytochrome P-450 in human placenta and lymphocytes.

A multiplicity of cytochromes P-450 is responsible for the detoxification and activation of xenobiotics such as drugs and carcinogens. Individual differences in sensitivity to these agents may reside in the cytochrome P-450 phenotype. A monoclonal antibody-directed radioimmunoassay was developed that detects epitope-specific cytochromes P-450 in human placentas and peripheral lymphocytes. Placentas from women who smoked cigarettes contained greater amounts of cytochrome P-450 with the monoclonal antibody-specific epitope than placentas from nonsmokers. The amount of this cytochrome P-450 in human peripheral lymphocytes increased after treatment of the mitogenized lymphocytes with the cytochrome P-450 inducer benz[a]anthracene.

Monoclonal antibody-directed analysis of cytochrome P-450-dependent monooxygenases and mutagen activation in the livers of DBA/2 and C57BL/6 mice.

Monoclonal antibodies (MAb 1-7-1 and Mab 2-66-3) specific for cytochrome P-450 (cyt. P-450) isozymes inhibited the metabolism of carcinogens, other xenobiotics, and endogenous compounds in two strains of mice. Postmitochondrial liver supernatant (S9) was prepared from untreated, 3-methylcholanthrene-treated, phenobarbital-treated, and pregnenolone 16 alpha-carbonitrile-treated C57BL/6 (B6) and DBA/2 (D2) mice. The modifying effect of two types of MAb to a 3-methylcholanthrene-induced cyt. P-450 and a phenobarbital-induced cyt. P-450 was investigated for: (a) S9-mediated mutagenicity of aflatoxin B1, benzo(a)pyrene 7,8-dihydrodiol, 2-acetylaminofluorene, and N-nitrosomorpholine in Salmonella typhimurium strains; and (b) the activity of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, aminopyrine N-demethylase, and testosterone 6 beta-, 7 alpha-, and 16 beta-hydroxylases. With certain S9s, MAb-1-7-1 inhibited only those cytochrome P-450 isozymes involved predominantly in activity of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and ethoxycoumarin O-deethylase and mutagenicity of 2-acetylaminofluorene and benzo(a)pyrene 7,8-dihydrodiol; MAb 2-66-3 inhibited only those involved in aminopyrine N-demethylase and testosterone 6 beta-, 7 alpha, and 16 beta-hydroxylase activity and aflatoxin B1 mutagenicity. Both Mab 1-7-1 and MAb 2-66-3 inhibited cytochrome P-450 isozyme(s) implicated predominantly in testosterone 7 alpha-hydroxylation in S9 from pregnenolone 16 alpha-carbonitrile-treated B6 mice. MAb 1-7-1 did not inhibit N-nitrosomorpholine mutagenicity and MAb 2-66-3 increased it by 2- to 6-fold depending on the source of S9. Using these MAbs, it is thus possible to identify the contribution of the epitope-defined single or class of cyt. P-450 to specific metabolic reactions in S9 from untreated and inducer-treated mice.

The human peroxisome proliferator-activated receptor alpha gene: identification and functional characterization of two natural allelic variants.

Peroxisome proliferator-activated receptor (PPAR)alpha-null mice have a defect in fatty acid metabolism but reproduce normally. The lack of a detrimental effect of the null phenotype in development and reproduction opens up the possibility for null or variant PPARalpha gene (PPARA) alleles in humans. To search the coding region and splice junctions for mutant and variant PPARalpha alleles, the human PPARalpha gene was cloned and characterized, and sequencing by polymerase chain reaction was carried out. Two point mutations in the human gene were found in the DNA binding domain at codons for amino acids 131 and 162. The allele containing the mutation in codon 162 (CTT to GTT, L162V) designated PPARA*3, was found at a high frequency in a Northern Indian population. Transfection assays of this mutant showed that the non-ligand dependent transactivation activity was less than one-half as active as the wild-type receptor. PPARA*3 was also unresponsive to low concentrations of ligand as compared to the wild-type PPARA*1 receptor. However, the difference is ligand concentration-dependent; at concentrations of the peroxisome proliferator Wy-14 643 > 25 microM, induction activity was restored in this variant's transactivation activity to a level five-fold greater as compared with wild-type PPARA*1 with no ligand. The mutation in codon 131 (CGA to CAA, R131Q), designated PPARA*2 is less frequent than PPARA*3, and the constitutive ligand independent activity was slightly higher than PPARA*1. Increasing concentrations of Wy-14 643 activated PPARA*2 similar to that observed with PPARA*1. The biological significance of these novel PPARalpha alleles remains to be established. It will be of great interest to determine whether these alleles are associated with differential response to fibrate therapy.

The effect of dilauroyl-L-3-phosphatidylcholine on the interaction between cytochrome P-450 1A1 and benzo[a]pyrene.

Fluorescence quenching of benzo[a]pyrene (BP) by cytochrome P-450 1A1 was used to probe the effect of the lipid, dilauroyl-L-3-phosphatidylcholine, on this substrate-enzyme interaction. In the presence of lipid, a monoclonal antibody to this P-450 maximally inhibited BP binding at an antibody-to-P-450 ratio of 1:2, corresponding to an antibody crosslinked P-450 complex. The antibody did not inhibit BP binding in the absence of lipid. These results indicate that when P-450 is subjected to the orientational constraints imposed by antibody-mediated crosslinking, the lipid alters the conformation or quaternary structure of the P-450 oligomer in a manner which changes its affinity for BP.

Specificity of the cytochrome P-450 interaction with cytochrome b5.

The specificity of the interaction of cytochrome b5 with different forms of cytochrome P-450 was examined. Immunopurification of cytochromes P-450 1A1, 2B1 and 2E1 from rat liver microsomes resulted in co-purification of cytochrome b5 with cytochrome P-450 forms 2B1 and 2E1 but not 1A1. This specificity was evaluated in conjunction with multiple sequence alignment of the three cytochrome P-450s and a molecular model of the cytochrome P-450-cytochrome b5 complex [(1989) Biochemistry 28, 8201-8205]. These analyses suggest two basic residues in the arginine cluster region of P-450, which are present in P-450s 2B1 and 2E1 but are absent in P-450 1A1, as potential binding sites for cytochrome b5.

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line.

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.

Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics.

The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.

A fluorescence study of the interactions of benzo[a]pyrene, cytochrome P450c and NADPH-cytochrome P450 reductase.

Fluorescence quenching of benzo[a]pyrene (BP) by cytochrome P450c was used to probe this substrate-enzyme binding interaction. Addition of NADPH-cytochrome P450 reductase, an essential electron carrier during P450 catalysis, resulted in increased quenching and thus strengthened binding of BP to P450c. This shows that the role of reductase extends beyond that of an electron transfer agent to influence substrate binding. Fluorescence titration measurements revealed that reductase and P450c formed a complex with an apparent KD of 13.7 +/- 0.9 nM. Reductase had no effect in the presence of an anti-P450c monoclonal antibody which inhibits BP hydroxylation, which suggests that this monoclonal antibody binds P450c near its reductase binding region.

Drug-drug interactions: effect of quinidine on nifedipine binding to human cytochrome P450 3A4.

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

Prevention of 2-acetylaminofluorene-induced loss of nuclear envelope cytochrome P450 by the simultaneous administration of 3-methylcholanthrene.

Rats fed a basal diet containing 0.05% (w/w) 2-acetylaminofluorene (AAF) for 3 weeks showed a 50% loss of hepatic nuclear envelope cytochrome P450, whereas microsomal P450 remained at control levels. A similar dietary treatment with 0.004% (w/w) 3-methylcholanthrene (MC) caused moderate losses (20-25%) of cytochrome P450 in both nuclear envelopes and microsomes. Administration of the basal diet supplemented with a mixture of AAF (0.05%) plus MC (0.004%) resulted in a preservation of control levels of nuclear envelope cytochrome P450 and a 30% elevation of microsomal P450. Immunoblot analysis revealed that AAF alone, or in concert with MC, induced comparable levels of the P450d form. Induction of cytochrome P450c by dietary MC was detected only when MC was fed together with AAF. As previously found for butylated hydroxytoluene (BHT), the protective effect of dietary MC against hepatocarcinogenesis in AAF-fed rats correlated with a preservation of nuclear envelope cytochrome P450 content and with the induction of cytochrome P450c.

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.

Induction of rat liver microsomal and nuclear cytochrome P-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene.

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.

Selective inhibition of oncogenic ras-p21 in vivo by agents that block its interaction with jun-N-kinase (JNK) and jun proteins. Implications for the design of selective chemotherapeutic agents.

We have obtained evidence that oncogenic and activated normal ras-p21 proteins utilize overlapping but distinct signal transduction pathways. Recently, we found that ras-p21 binds to both jun and its kinase, jun kinase (JNK). We now present evidence that suggests that oncogenic but not normal activated p21 depends strongly on early activation of JNK/jun. This early activation most likely involves direct interaction between oncogenic p21 and JNK/jun because p21 peptides that blocked the binding of p21 to JNK and jun strongly inhibited oncogenic p21-induced oocyte maturation while they did not inhibit insulin-activated normal cellular p21-induced maturation. Very similar results were also obtained for a newly characterized specific inhibitor of JNK which blocked oncogenic but not normal activated p21-induced oocyte maturation. We also found that both jun and JNK strongly enhanced oncogenic p21-induced oocyte maturation while they inhibited insulin-activated normal p21-induced oocyte maturation. These results suggest that the peptides and JNK inhibitor may be useful agents in selectively blocking the effects of oncogenic but not normal p21 in cells.

Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

PURPOSE: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. METHODS: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). RESULTS: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. CONCLUSION: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s.

Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7alpha-, 6beta- and 2alpha-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2'-isopropyloxy-4'-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.

Inhibition of methoxyresorufin demethylase activity by flavonoids in human liver microsomes.

Flavonoids are a class of dietary phytochemicals with anticarcinogenic properties. A series of ten structurally related flavonoids were evaluated for their effect on methoxyresorufin O-demethylase (MROD) activity in human liver microsomes. All compounds inhibited this cytochrome P450 1A2 (CYP1A2) mediated activity. 3,5,7-Trihydoxyflavone (galangin) was the most potent inhibitor, followed by 3-hydroxyflavone and flavone. The relative inhibitory potency of flavonoids is related to their structures. The results suggest that flavonoids may modulate pharmacological and toxicological effects mediated by CYP1A2.

Activated conformations of the ras-gene-encoded p21 protein. 2. Comparison of the computed and high-resolution x-ray crystallographic structures of Gly-12 p21.

The ras-oncogene-encoded p21 protein is a G-protein that has been shown to cause the malignant transformation of normal cells and has been implicated in causing human tumors. p21 is thought to be activated by the binding of GTP in place of GDP to the protein. We have previously constructed the three-dimensional structure of the p21 protein bound to GDP from an available alpha-carbon tracing of this protein using a combination of molecular dynamics and energy minimization (Dykes, et al., J. Biomol. Struct. Dynamics, 9:1025-1044). Until the recent publication of the all-heavy-atom x-ray crystallographic molecular coordinates of p21 residues 1-166 bound to a non-hydrolyzable GTP derivative (GppNp), no all-atom structure of the p21 protein has been available in the Brookhaven National Laboratories Protein Data Bank (PDB). In this communication we compare our computed structure for the p21-GDP complex to this x-ray crystal structure. We find that the two structures agree quite closely with one another, the overall RMS deviation for the backbone being 1.47 A and 2.71 A for all of the atoms. We have identified the regions of the protein that are responsible for the most significant deviations between the two structures, i.e., residues 32-40 and 61-74. We find that the main chain in the 32-40 segment deviates significantly from residue 32 to residue 36 and the side chain phenolic rings of residue 32 differ greatly between the two structures. The 61-74 region is the least-well defined region in the whole protein crystallographically having, by far, the highest temperature factor (B-factor). The backbone and side chain conformations in the 61-74 segment differ markedly, the overall RMS deviation being 3.1 A for the backbone and 5.7 A for all atoms. Both of these regions have been found in x-ray crystallographic studies of p21-GDP and p21-GTP complexes to undergo significant changes in conformation upon the binding of GTP in place of GDP to the protein. We have further compared our computed structure of the p21 protein with the x-ray crystal structure with regard to the conformations of individual segments, in particular, structurally conserved sequences (SCR), i.e., those sequences that have structural and sequence homology to corresponding sequences in the related G-protein, bacterial elongation factor Tu(EF-Tu), and variable loop regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular dynamics of the H-ras gene-encoded p21 protein; identification of flexible regions and possible effector domains.

We previously reported a complete computer-based three-dimensional structure for residues 1-171 of the Gly 12-containing ras-gene-encoded p21 protein complexed with GDP. This structure was subsequently shown to closely agree with a high-resolution x-ray crystallographic structure of p21. In this communication, we report a molecular dynamics stimulation of the modelled structure in an explicit shell of water molecules to identify domains within the protein that are unusually flexible. These domains represent regions which are most likely to undergo important conformational changes when the protein is activated by binding to GTP or by oncogenic amino acid substitutions such as Val for Gly 12. The starting structure was surrounded with water molecules, temperature-equilibrated and then followed over a 100 ps trajectory during which time the energy converged after about 50 ps. Regions of the protein that were found to have the largest coordinate fluctuations involved residues 12-16, 30-35, 40-52, 60-73, 85-89, 101-109, 119-123, and 127-131. Many of these sequences with high flexibility have been implicated in the functioning of this protein. Since the overall largest fluctuations were observed for residues 101-106 and 119-123, p21 peptides containing these residues (96-110 and 115-126) were synthesized and were found to inhibit strongly the effects of oncogenic p21 protein in an oocyte maturation assay. These results indicate that the flexible p21 sequences may constitute critical functional domains of the activated protein and that this general approach may be useful for identification of important functional domains in proteins.