Structure of A beta(25-35) peptide in different environments

The fragment A beta(25-35) of the Alzheimer's amyloid beta-peptide, like its full-length peptide A beta(1-42), has shown neurotoxic activities in cultured cells. The conformational preference of this important peptide is examined here in solution, gel, and film states (obtained with organic and aqueous solvents) by vibrational circular dichroism spectroscopy for the first time. For comparative studies, vibrational absorption and electronic circular dichroism measurements were also carried out under identical conditions. The peptide was found to adopt beta-sheet and beta-turn structures, with their relative proportions changing in different environments.     

Effect of D-amino acids at Asp23 and Ser26 residues on the conformational preference of Abeta20-29 peptides

The effects of d-amino acids at Asp(23) and Ser(26) residues on the conformational preference of beta-amyloid (Abeta) peptide fragment (Abeta(20-29)) have been studied using different spectroscopic techniques, namely vibrational circular dichroism (VCD), vibrational absorption, and electronic circular dichroism. To study the structure of the Abeta(20-29), [d-Asp(23)]Abeta(20-29), and [d-Ser(26)]Abeta(20-29) peptides under different conditions, the spectra were measured in 10mM acetate buffer (pH 3) and in 2,2,2-trifluoroethanol (TFE). The spectroscopic results indicated that at pH 3, Abeta(20-29) peptide takes random coil with beta-turn structure, while [d-Ser(26)]Abeta(20-29) peptide adopts significant amount of polyproline II (PPII) type structure along with beta-turn contribution and d-Asp-substituted peptide ([d-Asp(23)]Abeta(20-29)) adopts predominantly PPII type structure. The increased propensity for PPII conformation upon d-amino acid substitution, in acidic medium, has important biological implications. In TFE, Abeta(20-29), [d-Asp(23)]Abeta(20-29), and [d-Ser(26)]Abeta(20-29) peptides adopt 3(10)-helix, alpha-helix, and random coil with some beta-turn structures, respectively. The VCD data obtained for the Abeta peptide films suggested that the secondary structures for the peptide films are not the same as those for corresponding solution and are also different among the Abeta peptides studied here. This observation suggests that dehydration can have a significant influence on the structural preferences of these peptides.     

Glycosylation of synthetic peptides breaks helices. Phosphorylation results in distorted structure.

Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water-trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) beta-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function.     

Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor.

The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.     

Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.     

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclization of several linear penta- and heptapeptides with different metal ions studied by CD spectroscopy.

A cyclic pentapeptide c(Tyr-Leu-Ala-Gly-Pro) (I), which was isolated and identified from Pseudostellaria heterophylla medicinal herbs, and two cyclic heptapeptides, c(Gly-Tyr-Gly-Gly-Pro-Phe-Pro) (II) and c(Gly-Ile-Pro-Tyr-Ile-Ala-Ala) (III), which were isolated and identified from Stellaria yunnanensis Franch (M), were synthesized by using 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3 H)-one (DEPBT) as a coupling reagent in solution, and mediated by different metal ions, from their linear peptide precursors H-Tyr-Leu-Ala-Gly-Pro-OH (I-1) and H-Ala-Gly-Pro-Tyr-Leu-OH (I-2), H-Gly-Tyr-Gly-Gly-Pro-Phe-Pro-OH (II-1) and H-Gly-Ile-Pro-Tyr-Ile-Ala-Ala-OH (III-1), respectively. The results show that alkali metal ions can improve the cyclization yields and/or the cyclization rates of linear peptide precursors, such as Na(+) ion is favorable for the cyclization of linear pentapeptides and Cs(+) ion is favorable for the cyclization of linear heptapeptides, while some bivalent and trivalent metal ions, such as Mg(2+), Ca(2+), Zn(2+), Fe(2+), Ni(2+) and Cr(3+) reduced/inhibited both the cyclization yields and the cyclization rates of the linear peptide precursors. The circular dichroism spectra of I-1, II-1 and III-1 with different metal ions were studied to elucidate the changes in their secondary structures. It is shown that Cs(+) can induce and stabilize the type I beta-turn conformation in the linear heptapeptide II-1 and the type II beta-turn conformation in the linear heptapeptide III-1.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

The structure of antimicrobial pexiganan peptide in solution probed by Fourier transform infrared absorption, vibrational circular dichroism, and electronic circular dichroism spectroscopy

Pexiganan (Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys), a 22 amino acid peptide, is an analogue of the magainin family of antimicrobial peptides present in the skin of the African clawed frog. Conformational analysis of pexiganan was carried out in different solvent environments for the first time. Organic solvents, trifluoroethanol (TFE) and methanol, were used to study the secondary structural preferences of this peptide in the membrane-mimicking environments. In addition, aqueous (D2O) and dimethyl sulfoxide (DMSO) solutions were also investigated to study the role of hydrogen bonding involved in the secondary structure formation. Fourier transform infrared absorption, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) measurements were carried out under the same conditions to ascertain the conformational assignments in different solvents. All these spectroscopic measurements suggest that the pexiganan peptide has the tendency to adopt different structures in different environments. Pexiganan appears to adopt an alpha-helical conformation in TFE, a sheet-stabilized beta-turn structure in methanol, a random coil with beta-turn structure in D2O, and a solvated beta-turn structure in DMSO.     

Structure-function analysis of tritrypticin, an antibacterial peptide of innate immune origin.

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.     

Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides

Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized.     

Conformational changes in pediocin AcH upon vesicle binding and approximation of the membrane-bound structure in detergent micelles.

Pediocin AcH is a 44-residue antimicrobial peptide with bactericidal potency against Gram-positive bacteria such as Listeria. It belongs to a family of bacteriocins that, when membrane-associated, is predicted to contain beta-sheet and alpha-helical regions. All bacteriocins in this family have a conserved N-terminal disulfide bond. An additional C-terminal disulfide bond in pediocin AcH is thought to confer enhanced potency and broader specificity range against sensitive bacteria. The C-terminal disulfide bond may also affect the conformation of the C-terminus. The secondary structures of pediocin AcH in aqueous solution and vesicles from susceptible cells, as well as the ability of trifluoroethanol (TFE) and detergent systems to induce secondary structures like those induced in vesicles, were studied by circular dichroism (CD) spectroscopy. Like related peptides, pediocin AcH was highly unordered in aqueous solution, 56%. However, it also contained 20% beta-strand and 15% beta-turn structures. Upon complete binding to vesicles, 32% alpha-helical structure formed, the unordered structure decreased to 32%, and the beta-strand and beta-turn structures remained largely unchanged. Thus, a betaalpha domain structure formed in vesicles. The helical structure likely forces the C-terminal tail to loop back on the helix so that the C24-C44 disulfide bond can form. Detergent micelles were superior to TFE in their ability to induce secondary structural fractions in pediocin AcH comparable to those observed in vesicles. This demonstrates the importance of a hydrocarbon-water interface to pediocin AcH structure induction and suggests that it is preferable to use detergent micelles as solvents in NMR studies of pediocin AcH structure.     

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.     

Circular dichroism and fluorescence spectroscopic analyses of a proline-rich glycoprotein from human parotid saliva

A proline-rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda-max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30% beta-form conformation was predicted. Circular dichroism of PRG failed to detect either poly-L-proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, beta-turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of the N-linked Asn residues participating in some type of beta-turn.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

Design of peptides with branched beta-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3.

Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched beta-carbon residues are known to be different from other residues. As an implication it was expected that the branched beta-carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched beta-carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched beta-carbon residues induced beta-turn II conformation when placed at (i + 2) position while branched beta-carbon residues induced beta-turn III conformation. However, the conformation of a peptide with a nonbranched beta-carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched beta-carbon dehydro-residues instead of nonbranched beta-carbon residues and synthesized two peptides: (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3 with DeltaVal and DeltaIle, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II beta-turn conformations with torsion angles; (I): phi1 = -38.7 (4) degrees, psi1 = 126.0 (3) degrees; phi2 = 91.6 (3) degrees, psi2 = -9.5 (4) degrees and (II): phi1 = -37.0 (6) degrees, psi1 = 123.6 (4) degrees, phi2 = 93.4 (4), psi2 = -11.0(4) degrees respectively. Both peptide structures were stabilized by intramolecular 4-->1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1...O1' (-x, y + 1/2, -z) and N2...O2' (-x + 1, y + 1/2, -z) and van der Waals interactions.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

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.