Natural Abundance 13C NMR Research Articles

The effects of glycols on molecular mobility, structure, and permeability in stratum corneum

The skin provides an attractive alternative to the conventional drug administration routes. Still, it comes with challenges as the upper layer of the skin, the stratum corneum (SC), provides an efficient barrier against permeation of most compounds. One way to overcome the skin barrier is to apply chemical permeation enhancers, which can modify the SC structure. In this paper, we investigated the molecular effect of three different types of glycols in SC: dipropylene glycol (diPG), propylene glycol (PG), and butylene glycol (BG). The aim is to understand how these molecules influence the molecular mobility and structure of the SC components, and to relate the molecular effects to the efficiency of these molecules as permeation enhancers. We used complementary experimental techniques, including natural abundance 13C NMR spectroscopy and wide-angle X-ray diffraction to characterize the molecular consequences of these compounds at different doses in SC at 97% RH humidity and 32 °C. In addition, we study the permeation enhancing effects of the same glycols in comparable conditions using Raman spectroscopy. Based on the results from NMR, we conclude that all three glycols cause increased mobility in SC lipids, and that the addition of glycols has an effect on the keratin filaments in similar manner as Natural Moisturizing Factor (NMF). The highest mobility of both lipids and amino acids can be reached with BG, which is followed by PG. It is also shown that one reaches an apparent saturation level for all three chemicals in SC, after which increased addition of the compound does not lead to further increase in the mobility of SC lipids or protein components. The examination with Raman mapping show that BG and PG give a significant permeation enhancement as compared to SC without any added glycol at corresponding conditions. Finally, we observe a non-monotonic response in permeation enhancement with respect to the concentration of glycols, where the highest concentration does not give the highest permeation. This is explained by the dehydration effects at highest glycol concentrations. In summary, we find a good correlation between the molecular effects of glycols on the SC lipid and protein mobility, and macroscopic permeation enhances of the same molecules.

Low-temperature (

Electronic cigarette usage has spiked in popularity over recent years. The enhanced prevalence has consequently resulted in new health concerns associated with the use of these devices. Degradation of the liquids used in vaping have been identified as a concern due to the presence of toxic compounds such as aldehydes in the aerosols. Typically, such thermochemical conversions are reported to occur between 300 and 400 °C. Herein, the low-temperature thermal degradation of propylene glycol and glycerol constituents of e-cigarette vapors are explored for the first time by natural abundance 13C NMR and 1H NMR, enabling in situ detection of intact molecules from decomposition. The results demonstrate that the degradation of electronic nicotine delivery system (ENDS) liquids is strongly reliant upon the oxygen availability, both in the presence and absence of a material surface. When oxygen is available, propylene glycol and glycerol readily decompose at temperatures between 133 and 175 °C over an extended time period. Among the generated chemical species, formic and acrylic acids are observed which can negatively affect the kidneys and lungs of those who inhale the toxin during ENDS vapor inhalation. Further, the formation of hemi- and formal acetals is noted from both glycerol and propylene glycol, signifying the generation of both formaldehyde and acetaldehyde, highly toxic compounds, which, as a biocide, can lead to numerous health ailments. The results also reveal a retardation in decomposition rate when material surfaces are prevalent with no directly observed unique surface spectator or intermediate species as well as potentially slower conversions in mixtures of the two components. The generation of toxic species in ENDS liquids at low temperatures highlights the dangers of low-temperature ENDS use.

Anatomy of a selectively coassembled β-sheet peptide nanofiber

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar β-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled β-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer β-sheet amyloid structures predominate, but off-pathway β-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 μM. Natural abundance 13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, 13C-13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of β-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway β-barrel oligomers are also suggested to occur in coassembled β-strand peptide systems.

A metabolomics footprint approach to understanding the benefits of synbiotics in functional foods and dietary therapeutics for health, communicable and non-communicable diseases

Gut microbiota have been shown to affect various cellular and host response elements such as immunological, neurological, energy, storage, etc. In recent years, this has led to rapid expansion in dietary products containing probiotics, prebiotics and combination thereof in synbiotics. While benefits of consuming functional foods derived from probiotics strains have been demonstrated for various metabolites, a detailed analysis of the biochemical footprints and their benefits remain under-studied. Herein, using a combination of NMR metabolomics, microbial techniques and cell-culture assays, we have characterized metabolite profiles of probiotic viz. Lactobacillus delbruekii ATCC 9649, Lactobacillus casei ATCC 335, Lactobacillus plantarum NRC 716 and Bacillus coagulans ATCC 12425 cultures in fermented milk. We identified predominance of sugars, small chain fatty acids, organic acids and branched chain amino acids from natural abundance 13C NMR studies. Additionally, we identified myriad metabolites and their respective pathways using 1H NMR spectroscopy. Based on our findings, synbiotic fermented dairy products were customized with co-cultures and complemented with pro- and pre- biotics. Furthermore, we demonstrate epithelial cell interaction and anti-microbial activity of L. plantarum based ferment against a range of bacterial pathogens highlighting possible biochemical mechanisms for anti-microbial activity, quorum sensing, gut colonization and other beneficial factors that may be crucial. Furthermore, we propose plausible explanation against non-communicable diseases such as tumor-inhibitory, anti-proliferative and pro-apoptotic effects which has direct implications for dietary therapeutics.

Transferred Overhauser DNP: A Fast, Efficient Approach for Room Temperature 13C ODNP at Moderately Low Fields and Natural Abundance.

Overhauser dynamic nuclear polarization (ODNP) is investigated at a moderately low field (1.2 T) for natural abundance 13C NMR of small molecules in solution state at room temperature. It is shown that ODNP transferred from 1H to 13C by NMR coherence transfer is in general significantly more efficient than direct ODNP of 13C. Compared to direct 13C ODNP, we demonstrate over 4-fold higher 13C sensitivity (signal-to-noise ratio, SNR), achieved in one-eighth of the measurement time by transferred ODNP (t-ODNP). Compared to the 13C signal arising from Boltzmann equilibrium in a fixed measurement time, this is equivalent to about 1500-fold enhancement of 13C signal by t-ODNP, as against a direct 13C ODNP signal enhancement of about 45-fold, both at a moderate ESR saturation factor of about 0.25. This owes in part to the short polarization times characteristic of 1H. Typically, t-ODNP reflects the essentially uniform ODNP enhancements of all protons in a molecule. Although the purpose of this work is to establish the superiority of t-ODNP vis-à-vis direct 13C ODNP, a comparison is also made of the SNR in t-ODNP experiments with standard high resolution NMR as well. Finally, the potential of t-ODNP experiments for 2D heteronuclear correlation spectroscopy of small molecules is demonstrated in 2D 1H-13C HETCOR experiments at natural abundance, with decoupling in both dimensions.

Dynamic Nuclear Polarization NMR Spectroscopy Allows High-Throughput Characterization of Microporous Organic Polymers

Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance (13)C and (15)N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods.

The influence of thermochemical treatments on the lignocellulosic structure of wheat straw as studied by natural abundance 13C NMR

The effects of thermochemical treatments (aquathermolysis, pyrolysis, and combinations thereof) on the lignocellulosic structure and composition of wheat straw were studied with (13)C and (1)H solid state NMR spectroscopy and proton T1ρ relaxation measurements. Results show that aquathermolysis removes hemicellulose, acetyl groups, and ash minerals. As a result, the susceptibility of lignocellulose to pyrolysis is reduced most likely due to the removal of catalytically active salts, although recondensation of lignin during aquathermolysis treatment can also play a role. In contrast to pyrolysis of wheat straw, pyrolysis of aquathermolysed wheat straw leaves traces of cellulose in the char as well as more intense lignin methoxy peaks. Finally, it was found that both pyrolysis chars contain aliphatic chains, which were attributed to the presence of cutin or cutin-like materials, a macromolecule that covers the aerial surface of plants, not soluble in water and seemingly stable under the pyrolysis conditions applied.

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance 13C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance 13C NMR spectroscopy that provides complementary information to 2D 1H NMR. The correlation of 13Cα secondary shifts with both 3D structure and heteronuclear 15N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D 1H– 13Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. 1H– 13Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide–membrane interactions from different angles.

Fast enantioselective amino acid quantitative 13C NMR determination by a praseodymium chiral shift reagent

The fast quantitative determination of enantiomers of amino acids by the use of a praseodymium-based water-soluble chiral shift reagent, utilizing natural abundance 13C NMR spectra, is described.

High-Resolution Characterization of Liquid-Crystalline [60]Fullerenes Using Solid-State Nuclear Magnetic Resonance Spectroscopy

Liquid-crystalline materials containing fullerenes are valuable in the development of supramolecular switches and in solar cell technology. In this study, we characterize the liquid-crystalline and dynamic properties of fullerene-containing thermotropic compounds using solid-state natural abundance (13)C NMR experiments under stationary and magic angle spinning sample conditions. Chemical shifts spectra were measured in isotropic, liquid-crystalline nematic and smectic A and crystalline phases using one-dimensional (13)C experiments, while two-dimensional separated local-field experiments were used to measure the (1)H- (13)C dipolar couplings in mesophases. Chemical shift and dipolar coupling parameters were used to characterize the structure and dynamics of the liquid-crystalline dyads. NMR data of fullerene-containing thermotropic liquid crystals are compared to that of basic mesogenic unit and mesomorphic promoter compounds. Our NMR results suggest that the fullerene-ferrocene dyads form highly dynamic liquid-crystalline phases in which molecules rotate fast around the symmetry axis on the characteristic NMR time scale of approximately 10 (-4) s.

Adducts of zinc and copper(II) dimethyland diethyldithiocarbamates with piperidine: Synthesis, EPR, solid-state natural abundance 13C and 15N CP/MAS NMR and single-crystal X-ray diffraction studies

Crystalline adducts of zinc and copper(II) dimethyl-and diethyldithiocarbamates with piperidine (Pip) of the general formula [M{NH(CH2)5}(S2CNR2)2] (M = Zn and 63Cu; R = CH3 and C2H5) were obtained. Their structures and spectroscopic characteristics were studied by X-ray diffraction analysis, EPR spectroscopy, and solid-state natural abundance 13C and 15N MAS NMR spectroscopy. The most substantial differences between the adducts of the formula [Zn{NH(CH2)5}(S2CNR2)2] (R = CH3 and C2H5) were found in the spatial orientations of the coordinated heterocycles and the geometries of the zinc polyhedra. The individual character of the EPR spectra of magnetically diluted isotope-substituted copper(II) adducts was determined by computerassisted modeling. The adducts of copper(II) and zinc dimethyldithiocarbamates proved to exist as two isomers. The coordination polyhedra of copper(II) and zinc are intermediate between a tetragonal pyramid (TP) and a trigonal bipyramid (TBP). The contributions from the TBP/TP components to the coordination polyhedra were quantitatively estimated from X-ray diffraction data. The 13C and 15N NMR signals were assigned to the positions of the atoms of the =NC(S)S− groups in the resolved (according to X-ray diffraction data) molecular structures of the adducts.

NMR Investigation of the Electrostatic Effect in Binding of a Neuropeptide, Achatin-I, to Phosphatidylcholine Bilayers

Achatin-I (Gly1-d-Phe2-Ala3-Asp4), known as a neuropeptide containing a d-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+ (Kimura, T.; Okamura, E.; Matubayasi, N.; Asami, K.; Nakahara, M. Biophys. J. 2004, 87, 375-385). To gain mechanistic insights into how the binding equilibrium is delicately controlled by the ionization state of the N-terminal amino group, peptide-lipid binding interactions are investigated by selectively enriched 15N (at the N-terminus) and natural-abundance 13C NMR spectroscopy. Upon binding to the PC membrane, the 15N NMR of the N-terminal NH3(+) shifts upfield. This observation supports a mechanism that the role of the N-terminal NH3(+) in stabilizing the binding state is through electrostatic attraction with a headgroup negative charge, i.e., PO4(-). Interestingly, when the side chain beta-carboxyl group in Asp4 is deionized at acidic pH, the 15N signal of the N-terminal NH3(+) exhibits no significant chemical-shift change upon membrane binding of achatin-I. The Asp4 side chain thus regulates efficiency of the electrostatic binding between the peptide N-terminal NH3(+) and the lipid headgroup PO4(-). 13C chemical shifts in the hydrophobic D-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain beta-CO2(-) in Asp4 is deionized; the deionization of Asp4 beta-CO2(-) increases the net hydrophobicity of achatin-I with a reduction of both the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)3(+) in the most superficial region of the PC membrane, resulting in deeper anchoring of the phenyl ring. Hence, the electrostatic effect of the side chain beta-CO2(-) in Asp4 floats achatin-I on the PC membrane surface, and the binding equilibrium is sensitively controlled by the ionization state of the N-terminal NH3(+).

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance 13C and 14N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of 13C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using 14N experiments on bicelles is also discussed.

Chiral Phosphoramide-Catalyzed Aldol Additions of Ketone Trichlorosilyl Enolates. Mechanistic Aspects

The mechanism of the catalytic, enantioselective addition of trichlorosilyl enolates to aldehydes has been investigated. Kinetic studies using ReactIR and rapid injection NMR (RINMR) spectroscopy have confirmed the simultaneous operation of dual mechanistic pathways involving either one or two phosphoramides bound to a siliconium ion organizational center. This mechanistic dichotomy was initially postulated on the basis of catalyst loading studies and nonlinear effects studies. This duality explains the difference in reactivity and stereoselectivity of various classes of phosphoramides. Determination of Arrhenius activation parameters revealed that aldol addition occurs through the reversible albeit unfavorable formation of an activated complex, and natural-abundance 13C NMR kinetic isotope effect (KIE) studies have determined that the turnover limiting step is the aldol addition. A thorough examination of a range of phosphoramides has established empirical structure-activity selectivity relationships. In addition, the effects of catalyst loading, rate of addition, solvents, and additives have been studied and together allow the formulation of a unified mechanistic picture for the aldol addition.

Mobility and location of anesthetics in lipid bilayer membranes by high-resolution, high-field-gradient NMR

Natural-abundance 13C and high-field-gradient NMR at 600 MHz were applied to shed light on the binding site and the mobility of anesthetics in lipid bilayer membranes. A high-power pulsed-field-gradient probe was specially designed for the reliable monitoring of molecular motions in a viscous membrane environment. A newly developed sensitive probe for the 10 mm diameter tube was also used for the natural abundance 13C NMR measurement. Large unilamellar vesicles, rather than micelles, were adopted as the most appropriate model for cell membranes in view of the dynamical structure. The binding of ethanol, chloroform, and sevoflurane in membranes was not deep but shallow at the membrane interface whose polarity is intermediate between water and the hydrophobic core. The anesthetics moved much faster than the lipid matrices in membrane. The movement was in sharp contrast to the slow motion of endocrine disruptors, the mobility comparable with the membrane lipid diffusion.

Natural-abundance 13C nuclear-magnetic-resonance study of toxin II from Anemonia sulcata.

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved one-carbon and two-carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side-chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH-terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6 NH2-terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved carbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.

Natural-abundance 13C-NMR spectroscopy of two glyco-asparagines derived from the core of N-glycosidic carbohydrate chains.

13C-NMR spectroscopic studies were carried out on Manalpha(1->6)Man beta(1->4)GlcNAc beta(1->4)GlcNAc beta(1->N)-Asn and Man alpha(1->6)Man beta(1->4)GlcNAc beta(1->4)[Fuc alpha(1->6)]GlcNAc beta(1->N)Asn, which are part of the invariant core of N-glycosidically linked carbohydrates of glycoproteins. All resonances of proton-bearing carbon atoms were unambiguously assigned. Where possible, the assignments were confirmed by selective proton-decoupling experiments.

NMR Study on the Binding of Neuropeptide Achatin-I to Phospholipid Bilayer: The Equilibrium, Location, and Peptide Conformation

Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance 13C and high-resolution (of 0.01 Hz order) 1H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal N H 3 + group in achatin-I; the de-ionization of N H 3 + decreases the bound fraction of the peptide from ∼15% to nearly none. The electrostatic attraction between the N-terminal positive N H 3 + group and the negative P O 4 − group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the 13C chemical shifts ( δ) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Δ δ of the 13C resonance for almost all the carbon sites in achatin-I. The changes Δ δ for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic C O 2 − groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic D-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy.

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.