Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature

With the use of one-dimensional 1H nuclear magnetic resonance, two-dimensional correlated spectroscopy, and two-dimensional nuclear Overhauser enhancement spectroscopy, the exchange mechanisms for numerous individual amide protons in the basic pancreatic trypsin inhibitor (BPTI) were investigated over a wide range of p2H and temperature. Correlated exchange under an EX1 regime was observed only for the most slowly exchanging protons in the central hydrogen bonds of the antiparallel beta-sheet and only over a narrow range of temperature and p2H, i.e., above ca. 55 degrees C and between p2H 7 and 9, where the opening rates of the structure fluctuations which promote the exchange of these protons are of the order 0.1 min-1. At p2H below 7, the exchange of this most stable group of protons is uncorrelated and is governed by an EX2 mechanism. At p2H above 9, the exchange is also uncorrelated and occurs via either EX2 or EX1 processes promoted by strictly local structure fluctuations. For all other backbone amide protons in BPTI, the exchange was found to be uncorrelated and by an EX2 mechanism under all conditions of p2H and temperature where quantitative measurements could be obtained with the methods used, i.e., for kex approximately less than 5 min-1. From these observations with BPTI it can be concluded that the amide proton exchange in globular proteins is quite generally via EX2 processes, with rare exceptions for measurements with extremely stable protons at high temperature and basic p2H. This emphasizes the need for further development of suitable concepts for the structural interpretation of EX2 amide proton exchange [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57; Wagner, G., Stassinopoulou, C. I., & Wüthrich, K. (1984) Eur. J. Biochem. 145, 431-436] and for more detailed investigations of the intrinsic exchange rates for solvent-exposed amide protons in the "open" states of a protein [Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry (following paper in this issue)].     

Correlation between the amide proton exchange rates and the denaturation temperatures in globular proteins related to the basic pancreatic trypsin inhibitor.

Nuclear magnetic resonance was used to measure the hydrogen-deuterium exchange rates for individual interior amide protons in a group of small globular proteins related to the basic pancreatic trypsin inhibitor (BPTI). These proteins include two homologous proteins and seven chemical modifications of BPTI. It was previously shown that the spatial structure of BPTI is preserved in all these related proteins. The exchange rates for corresponding amide protons in the different proteins were found to vary by a factor of as much as 5 X 104. The proton exchange is correlated with the thermal stability of the proteins, i.e. the lower the denaturation temperature, the faster the NH exchange. Further evidence that the exchange of interior amide protons is promoted by global fluctuations of the protein structures comes from the observation that the order of the relative exchange rates for the individual protons is the same in all the different species. This is the third in a series of three papers on nuclear magnetic resonance studies of labile protons in BPTI-related proteins. A detailed interpretation of the data will be given in a forthcoming paper.     

On the pH dependence of amide proton exchange rates in proteins.

We have analyzed the pH dependencies of published amide proton exchange rates (kex) in three proteins: bovine pancreatic trypsin inhibitor (BPTI), bull seminal plasma proteinase inhibitor IIA (BUSI IIA), and calbindin D9K. The base-catalyzed exchange rate constants (kOH) of solvent exposed amides in BPTI are lower for residues with low peptide carbonyl exposure, showing that the environment around the carbonyl oxygen influences kOH. We also examined the possible importance of an exchange mechanism that involves formations of imidic acid intermediates along chains of hydrogen-bonded peptides in the three proteins. By invoking this "relayed imidic acid exchange mechanism," which should be essentially acid-catalyzed, we can explain the surprisingly high pHmin (the pH value at which kex reaches a minimum) found for the non-hydrogen-bonded amide protons in the beta-sheet in BPTI. The successive increase of pHmin along a chain of hydrogen-bonded peptides from the free amide to the free carbonyl, observed in BPTI, can be explained as an increasing contribution of the proposed mechanism in this direction of the chain. For BUSI IIA (pH 4-5) and calbindin D9K (pH 6-7) the majority of amide protons with negative pH dependence of kex are located in chains of hydrogen-bonded peptides; this situation is shown to be consistent with the proposed mechanism.     

Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE-derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root-mean-square deviation of 0.5 A for the well-defined region of the protein, residues 7-37. Many of the side-chains show now well-defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15-21 and 27-29. In solution, the six N-terminal and the two C-terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two-strand beta-sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7-12, adjacent to the beta-sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central beta-sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment-based programs for folding recognition, as opposite to disulfide-bond alignments, reveal new proteins of unrelated sequence and function with a similar structure.     

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.     

Structurally homologous toxins isolated from the Taiwan cobra (Naja naja atra) differ significantly in their structural stability

Cardiotoxin and neurotoxin analogues isolated from snake venom sources are highly homologous proteins (>50% homology) with similar three-dimensional structures but exhibit drastically different biological properties. In the present study, we compare the conformational stability of cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX), a neurotoxin analogue, from the Taiwan cobra (Naja naja atra), using circular dichroism spectroscopy and hydrogen-deuterium (H/D) exchange techniques in conjunction with two-dimensional NMR methods. Contrary to expectations, it is found that CTX III and CBTX differ significantly in their structural stabilities. The three-dimensional structure of CBTX is less stable than that of CTX III. The amide protons of residues at the N- and C-terminal ends of the CTX III molecule are strongly protected against H/D exchange, implying that the terminal ends of the molecule are bridged together by significant numbers of hydrogen bonds. However, in CBTX, amide protons at the terminal ends of the molecule do not exhibit an significant protection against H/D exchange. Comparison of the protection factors of the various amide protons in CTX III and CBTX reveals that the extraordinary stability of CTX III stems from the strong network of interactions among the residues at the N- and C-terminal ends and also due to the tight and ordered packing of the nonpolar residues involved in the triple-stranded, anti-parallel, beta-sheet segment of the molecule.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Mapping the core of the beta(2)-microglobulin amyloid fibril by H/D exchange

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.     

Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein

We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.     

Solution structure and internal dynamics of NSCP, a compact calcium-binding protein

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of N(H)-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.     

A stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.     

Synthesis and characterization of a beta-hairpin peptide that represents a 'core module' of bovine pancreatic trypsin inhibitor (BPTI)

A new strategy for the design and construction of peptide fragments that can achieve defined, nativelike secondary structure is presented. The strategy is based upon the hypothesis that 'core elements' of a protein, synthesized in a single polypeptide molecule, will favor nativelike structure, and that by incorporating a cross-link, nativelike core structure will dominate the ensemble as the more extended conformations are excluded. 'Core elements' are the elements of packed secondary structure that contain the slowest exchanging backbone amide protons in the native protein. The 'core elements' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-sheet (residues 18-24 and 29-35) and the small beta-bridge (residues 43-44). To test the design strategy, we synthesized an 'oxidized core module', which contains the antiparallel strands connected by a modified reverse turn (A27 replaced by D), a natural disulfide cross-link at the open end of the hairpin, and N- and C-termini blocking groups. A peptide with identical sequence but lacking the disulfide cross-link at the open end was used as the 'reduced core module' control. The conformational behavior of both peptides was examined using (1)H NMR spectroscopy. Chemical shift dispersion, chemical shift deviation from random coil values, sequential and long-range NOEs, and H/D amide exchange rates were compared for the two peptides. We conclude that the ensemble of oxidized and reduced core module conformations samples both nativelike 4:4 and non-native 3:5 beta-hairpin structure, and that the oxidized module samples nativelike structure for a greater fraction of the time than the reduced module.     

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for NH2 group rotation in buried asparagine side chains

Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.     

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution.

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by 1H NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35 degrees C, and pH 9.4 and 25 degrees C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or turn. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)