Class-dependent sequence alignment strategy improves the structural and functional modeling of P450s

Different procedures for obtaining homology models for P450s are investigated using various sequence alignments sharing various levels of sequence identity with available P450 crystal structures. In this analysis, we have investigated how well homology modeling can reproduce known crystal structures as well as how effectively these homology models can be used to reproduce known ligand-binding modes. Homology models obtained from sequence alignments that discriminate between Class I and Class II P450s are significantly closer to the experimental crystal structures and more closely reproduce known ligand's binding modes, than those obtained using sequence alignments that combine Class I and Class II P450s. The quality of the models is slightly improved by constructing hybrid-structure models that model three of the most variable regions of P450s independently from the rest of the protein: the B region that includes SRS1, the FG region that includes SRS2 and SRS3 and the beta4 region that includes SRS6.     

Cytochrome P450 active site plasticity: attenuation of imidazole binding in cytochrome P450(cam) by an L244A mutation

We have identified a P450(cam) mutation, L244A, that mitigates the affinity for imidazole and substituted imidazoles while maintaining a high affinity for the natural substrate camphor. The P450(cam) L244A crystal structure solved in the absence of any ligand reveals that the I-helix is displaced inwards by over 1 A in response to the cavity created by the change from leucine to alanine. Furthermore, the crystal structures of imidazole-bound P450(cam) and the 1-methylimidazole-bound P450(cam) L244A mutant reveal that the ligands have distinct binding modes in the two proteins. Whereas in wild-type P450(cam) the imidazole coordinates to the iron in an orientation roughly perpendicular to the plane of the heme, in the L244A mutant the rearranged I helix, and specifically residue Val247, forces the imidazole into an orientation almost parallel to the heme that impairs its ability to coordinate to the heme iron. As a result, the imidazole is much more weakly bound to the mutant than it is to the wild-type enzyme. Despite the constriction of the active site by the mutation, previous work with the L244A mutant has shown that it oxidizes larger substrates than the wild-type enzyme. This paradoxical situation, in which a mutation that nominally increases the active site cavity appears to decrease it, suggests that the mutation actually increases the active site maleability, allowing it to better expand to oxidize larger substrates.     

Comparison of the construction of a 3-D model for human thromboxane synthase using P450cam and BM-3 as templates: implications for the substrate binding pocket

A 3-D model of human thromboxane A₂ synthase (TXAS) was constructedusing a homology modeling approach based on information fromthe 2.0 crystal structure of the hemoprotein domains of cytochromeP450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenaseresembling eukaryotic microsomal cytochrome P450s in primarystructure and function. TXAS shares 26.4% residue identity and48.4% residue similarity with the P450BM-3 hemoprotein domain.The homology score between TXAS and P450BM-3 is much higherthan that between TXAS and P450cam. Alignment between TXAS andthe P450BM-3 hemoprotein domain or P450cam was determined throughsequence searches. The P450BM-3 or P450cam main-chain coordinateswere spplied to the TXAS main chain in those sements where thetwo sequences were well aligned. These segments were linkedto one another using a fragment search method, and the sidechains were added to produce a 3-D model for TXAS. A TXAS substrate,prostaglandin H₂ (PGH₂) was docked into the TXAS cavity correspondingto the arachidonic acid binding pocket in P450BM-3 or camphorbinding site in P450cam. Regions of the heme and putative PGH2binding cavities in the TXAS model were identified and analyzed.The segments and residues involved in the active-site pocketof the TXAS model provide reasonable candidates for TXAS proteinengineering and inhibitor design. Comparison of the TXAS modelbased on P450BM-3 with another TXAS model based on the P450BM-3with another TXAS model based on the P450cam structure indicatedthat P450BM-3 is a more suitable template for homology modelingof TXAS.     

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Site-directed mutants were constructed in cytochrome P-450_camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450_cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450_cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101M–T185F–V247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant.     

The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.     

Binding free energy calculations for P450cam-substrate complexes

A recently proposed semi-empirical method for calculating bindingfree energies was used to examine the binding of a variety ofsubstrates to cytochrome P450cam. For a set of 11 differentpotential substrates of cytochrome P450cam, both the absoluteand relative binding free energies were generally well reproduced.The mean error in the calculated absolute binding free energyfor all 11 compounds is 0.55 kcal/mol. Forty-eight out of 55calculated relative binding free energies have the correct signand the mean unsigned error between calculated and experimentalrelative binding free energies is 0.77 kcal/mol. For one substrate,thiocamphor, the effect of substrate orientation on the calculatedbinding free energy was examined. The ability of this methodto predict the effect of active site mutations was also examinedin two cases.     

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.     

Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression

Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2genes, display 88% amino acid sequence similarities. The dissimilaritiesof sequence between these two enzymes are primarily localizedwithin four discrete regions of the polypeptides that are separatedby regions of absolute sequence identity. IIA1 specificallyhydroxylates the prototype substrate testosterone at the 7 and6 position with a predominance of 7 metabolite. IIA2, on theother hand, hydroxylates this steroid at eight positions onthe molecule, with one of the most abundant metabolites being15hydroxytestosterone. To determine those amino acids responsiblefor the difference in testosterone hydroxylation specificities,chimeras were constructed between IIA1 and IIA2 cDNAs and expressedin cell culture using vaccinia-virus-mediated cDNA expression.Chimeras, in which the first 355 amino acids correspond to asingle enzyme, maintain the specificity associated with thatenzyme. Of six chimeras which have substitutions between aminoacids 161 and 276, two are inactive and the remaining four givesimilar metabolite profiles, in which both 7 and 15 hydroxylationspecificities have been lost. Two of these four chimeras arediametric apposites, suggesting that modification of eitherthe N-terminal or central regions of the enzymes results inconformational changes that prevent the specific binding interactionsresponsible for the narrow regioselectivity associated withIIA1 and 15-hydroxytestosterone formation associated with IIA2.     

Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure

Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min(-1)) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.     

Structural Diversification of Macrolactones by Substrate‐Flexible Cytochrome P450 Monooxygenases

The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C‐4 position, which is different from the intrinsic C‐12 hydroxylation position in the natural substrate. This is the first report of C‐4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14‐membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12‐membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post‐PKS oxidations.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 waspredicted based on the crystal coordinates of cytochrome P450camfrom Pseudomonas putida. As there was only 15% residue identitybetween the two enzymes, additional information was used toestablish an accurate sequence alignment that is a prerequisitefor model building. Twelve representative eukaryotic sequenceswere aligned and a net prediction of secondary structure wasmatched against the known -helices and ß-sheets ofP450cam. The cam secondary structure provided a fixed main-chainframework onto which loops of appropriate length from the humanP450IA1 structure were added. The model-built structure of thehuman cytochrome conformed to the requirements for the segregationof polar and nonpolar residues between the core and the surface.The first 44 residues of human cytochrome P450 could not bebuilt into the model and sequence analysis suggested that residues1–26 formed a single membrane-spanning segment. Examinationof the sequences of cytochrome P450s from distinct gene familiessuggested specific residues that could account for the differencesin substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene,was fitted into the proposed active site and this planar aromaticmolecule could be accommodated into the available cavity. Residuesthat are likely to interact with the haem were identified. Thesequence similarity between 59 eukaryotic enzymes was representedas a dendrogram that in general clustered according to genefamily. Until a crystallographic structure is available, thismodel-building study identifies potential residues in cytochromeP450s important in the function of these enzymes and these residuesare candidates for site-directed mutagenesis.     

Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type

Under anaerobic conditions, cytochromes P450 can reductivelydehalogenate heavily halogenated hydrocarbons, such as one-and two-carbon organic solvents. This catalytic capacity hasdrawn attention to the potential use of engineered forms ofP450s in the remediation of contaminated deep subsurface ecosystems.Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign,IL) and S.G.Sligar (personal communication) have observedrecentlythat an active-site variant of cytochrome P450cam (F87W) dechlorinatespentachloroethane approximately three times faster than thewild-type enzyme. Molecular dynamics simulations have revealedthat the mutant enzyme binding pocket remains smaller, and thatpentachloroethane assumes configurations closer to the heme-Fein the F87W mutant twice as often as in the wild-type enzyme.This result is consistent with a collisional model of dehalogenation,which agrees with experimental observations [Li and Wackett(1993) Biochemistry, 32, 9355–9361] that solutions containingwild-type P450cam dehalogenate pentachloroethane 100 times fasterthan those containing free heme. The simulations suggest thatit is unlikely that Trp87 significantly stabilizes the developingnegative charge on the substrate during carbon-halogen bondreduction. The design of improved microbiai enzymes that incorporateboth steric and electronic effects continues for use in remediatinghalogenated contaminants in situ     

Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.     

Construction and evaluation of a three-dimensional structure of cytochrome P450choP enzyme (CYP105C1)

The purpose of this work was to develop and carefully evaluateimproved strategies for constructing reliable 3-D models ofP450 isozymes. To this end, a unique combination of steps forbuilding and evaluating a model structure was used to builda homology model of the P450choP isozyme, based on knowledgeof the X-ray structures of P450cam, P450terp, P450BM-3 and P450eryF.Specifically, the reliability of this model was examined bysystematic comparisons of its conformational, energetic, environmentaland packing properties and those of the four reference proteinswith corresponding properties from the database of proteinswith known structures. The results showed that the examinedproperties of this model structure are well within the criteriaestablished for reliable structures and are of nearly as goodquality as those of the reference proteins. In addition, theresult from a 120 ps unconstrained MD simulation of the modelwith structural waters provided evidence that the model is stableat room temperature. This 3-D model can now be reliably usedfor explicit characterization of substrate and inhibitor complexes.Most importantly, although it is envisioned that building modelsfor mammalian P450s will be even more challenging, the stepsdescribed here should be very useful in future constructionof 3-D models of mammalian P450 isozymes.     

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.     

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins

The DNA sequence of bovine microsomal cytochrome b₅ has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome b_s in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b₅ cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D ¹HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b₅. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (T_m of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (T_m 78°C) reflected in the values of H_m, S_mand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome b_s is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.     

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.     

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.     

Challenges in capturing oxygenase activity in vitro

Biocatalysis using oxygenase or desaturase enzymes has the potential to add value to native fats and oils by adding oxygen, hydroxyl groups, or double bonds to create regio- and/or stereospecific products. These enzymes are a subset of the large class of oxidoreductase enzymes (from EC subgroups 1.13 and 1.14) involved with biological oxidation and reduction. In vitro biocatalytic processing using these enzymes is hampered by the high cost of the stoichiometric cofactors. This article reviews recent progress in developing in vitro redox enzyme biocatalysis for commercial-scale syntheses. Coenzyme recycling and electrochemical redox cycling as methods for cofactor regeneration are described and commercial applications indicated. Direct charge transfer without use of mediators is described as the cleanest way of introducing the reducing power into the catalytic cycle. Our electrochemically driven cytochrome P450_cam bioreactor is discussed as an example of direct charge transfer to a redox protein. Site-directed mutagenesis in the active site of the P450_cam monooxygenase greatly improved performance for the conversion of the nonnative substrate, styrene to styrene oxide. This epoxidation reaction was also shown to give a single product (styrene oxide) in the bioelectrochemical reactor when the diatomic oxygen co-substrate was managed properly. Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the material or equipment is necessarily the best available for the purpose.