Chemical Exchange Rates Research Articles

Study of Structural Stability of Cyclophilin A by NMR and Circular Dichroism Spectra

AbstractThe structural stability of cyclophilin A (CypA) was investigated using H/D exchange and temperature coefficients of chemical shifts of amide protons, monitored by 2D heteronuclear NMR spectroscopy. Amide proton exchange rates were measured by H/D exchange experiments for slow‐exchange protons and measured by SEA (Solvent Exposed Amides)‐HSQC experiments for fast‐exchange protons. Temperature coefficients of chemical shifts and hydrogen exchange rates of amide protons show reasonably good correlation with the protein structure. Totally, 44 out of 153 non‐proline assigned residues still exist in 86 d of hydrogen‐deuterium exchange at 4 °C, suggesting that CypA structure should be highly stable. Residues in secondary structures of α2, β1, β2, β5, β6 and β7 might constitute the hydrophobic core of the protein. The change in free energy of unfolding (ΔGu) of CypA was estimated to be (21.99±1.53) kJ·mol−1 by circular dichroism (CD). The large free energy change is also an indicator of the high structural stability.

Hyaluronan: the absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models.

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Determining chemical exchange rates of the uracil labile protons by NMR diffusion experiments

The exchange rates of the amido-protons of uracil with water were determined by NMR diffusion experiments and the results showed a factor 2 difference in lability between them, which was confirmed by more classical 2D-NMR exchange experiments.

Water partitioning in “dry” poly(ethylene oxide) alcohol (C mE n) nonionic surfactant—a proton NMR study

A proton NMR titration study is presented where in small increments quantities of water were added to “dry” C m E n nonionic surfactant. For a particular range of compositions, two resonances for the water/hydroxyl protons were observed that display large chemical shift increases as water content is increased indicating that water must partition between two chemical environments with a surprisingly slow chemical exchange rate. A detailed mechanism of how the increasing amounts of water are incorporated into the surfactant medium is presented accounting for all observed spectral changes.

Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched‐flow setup

Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor sigma32 free in solution. Our results indicate that the C-terminal sigma4 domain of sigma32, which is responsible for the recognition of the -35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous sigma-factor sigmaA in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.

Water T2 relaxation in sugar solutions

1H spin–spin relaxation times of water were measured with the CPMG sequence in dilute aqueous solutions of glucitol, mannitol, glycerol, glycol, the methyl d-pyranosides of α-glucose, β-glucose, α-galactose, β-galactose, α-xylose, β-xylose, β-arabinose and sucrose, α,α-trehalose, β-maltose, maltotriose and maltoheptaose. The relaxation-time dispersion was measured by varying the CPMG pulse spacing, τ. These data were interpreted by means of the Carver–Richards model in which exchange between water protons and labile solute hydroxyl protons provides a significant contribution to the relaxation. From the dependences on temperature and τ, parameters characteristic of the pool of hydroxyls belonging to a given solute were extracted by nonlinear regression, including: the fraction of exchangeable protons, P, the chemical-shift difference between water protons and hydroxyl protons, δ ω, the intrinsic spin–spin relaxation time, T 2, and the chemical exchange rate, k. These solute-specific parameters are related, respectively, to the concentration, identity, mobility and exchange life-time of the hydroxyl site. At 298 K, values of δ ω, T 2 and k were found to be of the order of 1 ppm, 100 ms and 1000 s −1, respectively. Effects of molecular size, conformation and solute concentration were investigated. The exchange mechanism was characterised by Eyring activation enthalpies and entropies with values in the ranges 50–70 kJ mol −1 and −10 to 60 J K −1 mol −1, respectively.

Theoretical analysis of water 1H T2 based on chemical exchange and polysaccharide mobility during gelation

It was demonstrated that macroscopic structural changes of polysaccharide chains, such as random coil–helix transition and aggregation of helices, accompanied with the sol–gel and the gel–sol transition can be monitored by numerically analyzing the temperature dependence of the observed water proton spin–spin relaxation time ( T 2obs) modified by the chemical exchange between water proton and labile proton on the chain. According to the numerical analysis of T 2obs, typical profiles of T 2obs against the temperature were simulated with various parameters of the relaxation time ( T 2 i ) and the mean residence time ( τ i ) of water and the labile protons on the random coil and the ordered chain as well as the activation energy ( E i , E ex, i ) for the motion and the chemical exchange of the respective proton. The difference of structural change of kappa- , iota- , and lambda-carrageenan aqueous systems in the cooling and the heating process was analyzed. In conclusion, (1) the characteristic profile of the temperature dependence of T 2obs, commonly observed in the temperature-induced gelling process, is attributable to the variation of fraction of the ordered chain and the relative rate of chemical exchange to the relaxation rate of labile protons on polysaccharide (1/ T 2p); (2) the loose aggregated or associated structure is detected for iota-carrageenan system; and (3) the thermally stable junction zones formed by well aggregated helices of kappa-carrageenan in the gel state provokes a thermal hysteresis in the temperature-induced structural change (experimentally, in T 2obs). It is clarified by the numerical simulation that the aggregated helices formed during further cooling down to the temperature below T sg results in the T 2obs shifts to a longer value in the heating process as compared with that in the cooling process.

Two Homologous Rat Cellular Retinol-binding Proteins Differ in Local Conformational Flexibility

Cellular retinol-binding protein I (CRBP I) and cellular retinol-binding protein II (CRBP II) are closely homologous proteins that play distinct roles in the maintenance of vitamin A homeostasis. The solution structure and dynamics of CRBP I and CRBP II were compared by multidimensional NMR techniques. These studies indicated that differences in the mean backbone structures of CRBP I and CRBP II were localized primarily to the αII helix. Intraligand NOE cross-peaks were detected for the hydroxyl proton in the NOESY spectrum of CRBP I-bound retinol, but not for CRBP II-bound retinol, indicating that the conformational dynamics of retinol binding are different for these two proteins. As determined by Lipari–Szabo formalism, both the apo and holo forms of CRBP I and CRBP II are conformationally rigid on the pico- to nanosecond timescale. transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill -based 15N relaxation dispersion experiments at both 500 MHz and 600 MHz magnetic fields revealed that 84 and 62 residues for apo-CRBP I and II, respectively, showed detectable conformational exchange on a micro- to millisecond timescale, in contrast to three and seven residues for holo-CRBP I and II, respectively. Thus binding of retinol markedly reduced conformational flexibility in both CRBP I and CRBP II on the micro- to millisecond timescale. The 15N relaxation dispersion curves of apo-CRBP I and II were fit to a two-state conformational exchange model by a global iterative fitting process and by an individual (residue) fitting process. In the process of carrying out the global fit, more than half of the residue sites were eliminated. The individual chemical exchange rates k ex, and chemical shift differences, Δδ, were increased in the putative portal region (αII helix and βC-βD turn) of apo-CRBP II compared to apo-CRBP I. These differences in conformational flexibility likely contribute to differences in how CRBP I and CRBP II interact with ligands, membranes and retinoid metabolizing enzymes.

1H NMR studies of maltose, maltoheptaose, alpha-, beta-, and gamma-cyclodextrins, and complexes in aqueous solutions with hydroxy protons as structural probes.

The (1)H NMR chemical shifts, coupling constants, temperature coefficients, and exchange rates have been measured for the hydroxy protons of aqueous solutions of alpha-, beta-, and gamma-cyclodextrins, maltose, and maltoheptaose. In cyclodextrins (CDs), the high chemical shift of the O(3)H signal and its small (3)J(OH,CH) value suggest that O(3)H is involved in a hydrogen bond. The small temperature coefficients and rate of exchange values of O(2)H and O(3)H confirm the involvement of O(3)H in hydrogen bonding and indicate that O(2)H is the hydrogen bond partner. In maltose, two distinct NMR signals with two different vicinal coupling constants are found for O(2')H. A cross-peak in the ROESY spectrum indicates chemical exchange between the O(2')H and O(3)H protons. The existence of two distinct NMR signals with different J values for O(2')H shows the influence of anomeric configuration on the O(2')H-O(3)H interaction. The effect of complexation with methyl benzoate, adamantane-1-carboxylic acid, adamantane-1-ol, and l- and d-tryptophane on the NMR spectra of the hydroxy protons of alpha-, beta-, and gamma-cyclodextrins and of maltose has been investigated. No significant spectral changes were observed upon addition of methyl benzoate and adamantane-1-carboxylic acid. The addition of adamantane-1-ol resulted in an upfield shift and a strong broadening of the O(2)H signal from alpha-CD, and a small temperature coefficient was measured upon complexation. The O(2)H and O(3)H signals in beta-CD were broadened and shifted downfield upon addition of l- and d-tryptophane.

The dynamic behavior of CheW from Thermotoga maritima in solution, as determined by nuclear magnetic resonance: implications for potential protein–protein interaction sites

Measurements of the 15N relaxation parameters have been used to characterize the backbone dynamics of CheW from Thermotoga maritima. The dynamic nature of residues that appeared disordered in our recent solution structure of CheW is confirmed by these dynamics measurements. We have interpreted the data in terms of the Lipari and Szabo ‘model-free’ approach. The derived order parameter, S 2, the { 1H}– 15N heteronuclear nuclear Overhauser effect (NOE) values, the chemical exchange rate, R ex, and the internal correlation time, τ e, show that CheW exhibits considerable motional freedom from the picosecond to millisecond time scales. These regions of the protein cluster within the framework of the three-dimensional structure and may indicate possible binding sites for other protein components of the bacterial chemotaxis receptor-signaling complex. The structure of CheW consists of two five-stranded β-barrel domains that pack together with an extensive hydrophobic core between the domains. Regions highlighted by dynamics measurements co-localize to specific regions of the three-dimensional structure of CheW previously implicated in the formation of bacterial chemotaxis receptor signaling complex. The motional properties of domain 2 of CheW suggest that this domain may be able to experience structural rearrangements that allow the exposure of a hydrophobic surface area that could be used as a binding surface for the other members of the receptor complex. Residues within domain 2 have been implicated in binding interactions for two chemotaxis proteins, CheA and the receptor. We further propose that domain 1 interacts with other components of the chemotaxis machinery, such as CheZ, or in the formation of clusters of signaling components.

Hydrogen exchange rates in proteins from water (1)H transverse magnetic relaxation.

Access to the fast exchange kinetics of labile protein hydrogens in solution is provided by exchange broadening of the water 1H NMR line. We analyzed the chemical shift modulation contribution of labile hydrogens in bovine pancreatic trypsin inhibitor (BPTI) to the transverse 1H spin relaxation rate, R2, of the bulk solvent. Both the experimental pH dependence and the CPMG dispersion of R2 could be quantitatively accounted for on the basis of known chemical shifts, exchange rates, and ionization constants for BPTI. This analysis provided, for the first time, the hydrogen exchange rate constants for Lys and Arg side chains in a protein and pointed to an internal catalysis of the N-terminal amino protons in BPTI by a salt bridge. The method can be used for mapping the hydrogen exchange rates in protein solutions and biomaterials, which may be important for the control of relaxation-weighted contrast in biological MRI.

Evidence for flexibility in the function of ribonuclease A.

The dynamic properties of the enzyme ribonuclease A (RNase A) were investigated through the use of solution NMR spin relaxation experiments. As determined by "model-free" analysis, RNase A is conformationally rigid on time scales faster than overall rotational tumbling (picoseconds to nanoseconds). The average order parameter, S(2), for RNase A is 0.910 +/- 0.051. However, 28 of the amino acid residues in RNase A were identified as undergoing chemical exchange on the microsecond to millisecond time scale. For 16 of these residues the microscopic chemical exchange rates, k(ex), were quantitated through the use of the relaxation-compensated CPMG (rcCPMG) experiment. The value of k(ex) was identical for all residues with an average of 1640 s(-1) and is similar to the RNase A k(cat) value of 1900 s(-1). Many of these mobile residues localize to the active site in RNase A and include the catalytically crucial amino acids His119 and Asp121. Additional motion is found in the B1, B2, and P0 subsites, suggesting a coupling of motion between the binding and catalytic sites. The activation energy of the observed millisecond motion was measured by applying the rcCPMG experiment at temperatures of 283, 293, and 298 K and was determined to vary between 3.6 and 7.4 kcal/mol. The measured barrier to conformational motion is similar to the activation barrier for the RNase A catalyzed reaction and thus would not be thermodynamically limiting to catalysis. These studies suggest a correlation of conformational exchange kinetics and thermodynamics derived from NMR measurements with those determined by biochemical means and are suggestive of an important role for flexibility in enzyme function.

Thermodynamic and Kinetic Approach of the Reactivity in Micellar Media. Reaction of 1,3,5-Trinitrobenzene upon Hydroxide Ion in Aqueous Solutions of Cationic Surfactants

The kinetics of the reaction of OH- with 1,3,5-trinitrobenzene in the presence of dodecyl and hexadecyltrimethylammonium bromide have been studied. The micellar effects on this reaction have been modelized with the help of a new thermodynamic and kinetic analysis. This modelization has been based on a profitable approach proposed by De Donder and Defay which is well suited to the kinetics in dispersed medium. The chemical exchange rates σv0 and bv0, which are, respectively, the micellar and the bulk exchange rates, have been determined. This report also reveals that the acceleration of chemical reactions is mainly due not only to a large increase of the chemical exchange rate on the micellar aggregates but also to the high stabilization of the activated complex.

1H NMR studies of hydroxy protons of the V[β-Gal(1→3)-α-GalNAc(1→O)]THPGY glycopeptide

The hydroxy protons of the disaccharide moiety in the glycopeptide Val-[β-Gal(1→3)-α-GalNAc(1→O)]-Thr-His-Pro-Gly-Tyr ( 1) have been investigated in aqueous solution using 1H NMR spectroscopy. The chemical shifts ( δ), coupling constants ( 3 J CH,OH), temperature coefficients (d δ/d T), exchange rates ( k ex), and NOEs have been measured. The data show that the O(2′)H of Gal has a reduced contact with water due to steric interference caused by the 2-acetamido group of GalNAc. No interaction, in terms of hydrogen bonding exists between the disaccharide and the peptide moieties, but the rotation around the sugar-peptide linkage is restricted.

Studies of magnetization transfer and relaxation in irradiatedpolymer gels - interpretation of MRI-based dosimetry

Magnetization transfer and NMR relaxation rates were measured for waterprotons in two types of polymer gels developed for radiation dosimetry withMRI in order to quantify the contributions of different relaxationprocesses to the radiation response in such gels. Measurements included therate of magnetization transfer between proton pools and the ratio of thesizes of exchanging pools, R1 and R2. A model of relaxation inirradiated gels is presented to explain their properties. The modelincorporates three proton pools: free water, macromolecular andinterfacial. Two pools are insufficient to model the data. In thesesystems, radiation-induced polymerization appears to increase the size of asolid-like macromolecular proton pool but does not affect the rate constantof magnetization transfer per proton from macromolecular protons to thefree water protons. The relation between R1 and the pool size ratio isconsistent with free water exchanging with a macromolecular pool with anR1 of ~8 Hz. In addition, the rate of magnetization transfer isnot limited by the rate of chemical exchange between the free water and theinterfacial protons, and magnetization transfer most probably occurs vialabile proton exchange rather than via bound water molecules.

On neglecting chemical exchange effects when correcting in vivo (31)P MRS data for partial saturation.

Signal acquisition in most MRS experiments requires a correction for partial saturation that is commonly based on a single exponential model for T(1) that ignores effects of chemical exchange. We evaluated the errors in (31)P MRS measurements introduced by this approximation in two-, three-, and four-site chemical exchange models under a range of flip-angles and pulse sequence repetition times (T(R)) that provide near-optimum signal-to-noise ratio (SNR). In two-site exchange, such as the creatine-kinase reaction involving phosphocreatine (PCr) and gamma-ATP in human skeletal and cardiac muscle, errors in saturation factors were determined for the progressive saturation method and the dual-angle method of measuring T(1). The analysis shows that these errors are negligible for the progressive saturation method if the observed T(1) is derived from a three-parameter fit of the data. When T(1) is measured with the dual-angle method, errors in saturation factors are less than 5% for all conceivable values of the chemical exchange rate and flip-angles that deliver useful SNR per unit time over the range T(1)/5 < or = T(R) < or = 2T(1). Errors are also less than 5% for three- and four-site exchange when T(R) > or = T(1)(*)/2, the so-called "intrinsic" T(1)'s of the metabolites. The effect of changing metabolite concentrations and chemical exchange rates on observed T(1)'s and saturation corrections was also examined with a three-site chemical exchange model involving ATP, PCr, and inorganic phosphate in skeletal muscle undergoing up to 95% PCr depletion. Although the observed T(1)'s were dependent on metabolite concentrations, errors in saturation corrections for T(R) = 2 s could be kept within 5% for all exchanging metabolites using a simple interpolation of two dual-angle T(1) measurements performed at the start and end of the experiment. Thus, the single-exponential model appears to be reasonably accurate for correcting (31)P MRS data for partial saturation in the presence of chemical exchange. Even in systems where metabolite concentrations change, accurate saturation corrections are possible without much loss in SNR.

Rate constants for chemical exchange between water and hydroxyl protons in natural compounds evaluated with NMR two-dimensional exchange spectroscopy and geometric average method

Nuclear magnetic resonance two-dimensional exchange spectroscopy has been applied to measure the chemical exchange rate constants between water and hydroxyl protons in three natural organic compounds which contain four to eight hydroxyl groups. In data treatment, the simple geometric average method has been used. Application of the geometric average method to assumed exchange systems has indicated that the method is reliable.

Solution Structure of O-glycosylated C-terminal Leucine Zipper Domain of Human Salivary Mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-β-(1–3)-α-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nα-Fmoc-Ser-[Ac4-β -D-Gal-(1,3)-Ac2-a-D-GalN3]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a β-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CαH chemical shift perturbations, 3JNH-cαH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts α-helical structure from Ile2-Val21 and the glycopeptide II adapts α-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.

Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem. 274, 16681-16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85-93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1-211), troponin I-(33-211), troponin I-(1-80) and bisphosphorylated troponin I-(1-80). The cardiac specific N terminus, residues 1-32, and the C-domain, residues 81-211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: implications for drug transport.

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R(1)-D-Xaa-D-Phe-NHMe) homologues were each dissolved in a micellar d(38)-dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen-bond potential, and charge were either tightly controlled or well-characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide-NH-H(2)O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with (3)J(HH)-derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that-other factors being equal-flexible dipeptides having a propensity to fold together nonpolar N- and C-terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.