Structure of human insulin monomer in water/acetonitrile solution

Here we present evidence that in water/acetonitrile solvent detailed structural and dynamic information can be obtained for important proteins that are naturally present as oligomers under native conditions. An NMR-derived human insulin monomer structure in H₂O/CD₃CN, 65/35 vol%, pH 3.6 is presented and compared with the available X-ray structure of a monomer that forms part of a hexamer (Acta Crystallogr. 2003 Sec. D59, 474) and with NMR structures in water and organic cosolvent. Detailed analysis using PFGSE NMR, temperature-dependent NMR, dilution experiments and CSI proves that the structure is monomeric in the concentration and temperature ranges 0.1–3 mM and 10–30°C, respectively. The presence of long-range interstrand NOEs, as found in the crystal structure of the monomer, provides the evidence for conservation of the tertiary structure. Starting from structures calculated by the program CYANA, two different molecular dynamics simulated annealing refinement protocols were applied, either using the program AMBER in vacuum (AMBER_VC), or including a generalized Born solvent model (AMBER_GB). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wojciech Bocian contributed equally to this work.     

Novel recombinant insulin analogue with flexible C-terminus in B chain. NMR structure of biosynthetic engineered A22G-B31K-B32R human insulin monomer in water/acetonitrile solution

A tertiary structure of recombinant A22^G-B31^K-B32^R-human insulin monomer (insulin GKR) has been characterized by ¹H, ¹³C NMR at natural isotopic abundance using NOESY, TOCSY, ¹H/¹³C-GHSQC, and ¹H/¹³C-GHSQC-TOCSY spectra. Translational diffusion studies indicate the monomer structure in water/acetonitrile (65/35 vol.%). CSI analysis confirms existence of secondary structure motifs present in human insulin standard (HIS). Both techniques allow to establish that in this solvent recombinant insulin GKR exists as a monomer. Starting from structures calculated by the program CYANA, two different refinement protocols used molecular dynamics simulated annealing with the program AMBER; in vacuum (AMBER_VC), and including a generalized Born solvent model (AMBER_GB). From these calculations an ensemble of 20 structures of lowest energy was chosen which represents the tertiary structure of studied insulin. Here we present novel insulin with added A22^G amino acid which interacts with β-turn environment resulting in high flexibility of B chain C-terminus.     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Self-assembling peptides: sequence, secondary structure in solution and film formation

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable beta-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce beta-sheet and thus film formation and that conformations different from beta-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 906-915, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

A refined model for the solution structure of oxidized putidaredoxin

A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry ) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.     

Conformational studies of the alpha-helical 28-43 fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

To determine whether the alpha-helix in the B3 immunoglobulin binding domain of protein G from group G Streptococcus has conformational stability as an isolated fragment, we carried out a CD and NMR study of the 16-residue peptide in solution corresponding to this alpha-helix. Based on two-dimensional H-NMR spectra recorded at three different temperatures (283, 305, and 313 K), it was found that this peptide is mostly unstructured in water at these temperatures. Weak signals corresponding to i,i+3 or i,i+4 interactions, which are characteristic of formation of turn-like structures, were observed in the ROE spectra at all temperatures. The absence of a stable three-dimensional structure of the investigated peptide supports an earlier study (Blanco and Serrano, Eur J Biochem 1995, 230, 634-649) of a possible mechanism for folding of other (B1 and B2) immunoglobulin binding domains of Protein G. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1032-1044, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.     

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C==C stretching modes. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1045-1053, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Human tropoelastin sequence: Dynamics of polypeptide coded by exon 6 in solution

Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2‐trifluoroethanol (TFE), a structure‐inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent–peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with additionof TFE. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 943–952, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three alpha-helices and B-chain beta-turn) is similar to that observed in the 2-Zn crystal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structures. However, differences between insulin and DPI are observed in the extent of conformational broadening of amide resonances, indicating that the presence or absence of residues B26-B30 influences the overall dynamics of the protein on the millisecond time scale. (3) Residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. This configuration differs from that described in a more organic milieu (35% acetonitrile; Kline, A.D., & Justice, R.M., Jr. (1990) Biochemistry 29, 2906-2913), suggesting that the conformation of insulin in the latter study may have been influenced by solvent composition. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To our knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening. Such an analysis is made possible in the present case by comparative study of an analogue (DPI) with more tractable spectroscopic properties.     

Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics

The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25----Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.     

Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.     

Solvent interactions with the Trp-cage peptide in 35% ethanol-water

Intermolecular NOE experiments have been used to explore interactions of water and ethanol molecules in 35% ethanol/65% water (v/v) with the peptide Trp-cage at temperatures from 5 to 25 degrees C. Magnetic dipole-dipole cross-relaxation terms sigma(HH) (NOE) and sigma(HH) (ROE) for interaction of solvent components with spins of the peptide suggest that ethanol molecules associate with backbone atoms for times of the order of nanoseconds at 5 degrees C. Formation of peptide-ethanol complexes can also account for the larger-than-expected values of cross-relaxation terms at higher temperatures. Hydrocarbon side chains of the peptide do not appear to experience such interactions with ethanol. Cross relaxation resulting from water-peptide interactions are consistent with long-lived water interactions with the backbone atoms. Water cross relaxation with nonpolar side chains of the peptide (Leu2, Ile4, Leu7, and proline residues) are only those expected for bulk solvent. However, long-lived association of both water and ethanol with the polar side chains of Tyr3 and Trp6 is indicated by the data. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 862-872, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Secondary structure of pig skin proteodermatan sulfate: a perspective from Raman spectroscopic studies in aqueous solution

Raman spectroscopic studies of pig skin proteodermatan sulfate in H2O are indicative of a well defined secondary structure consisting of alpha-helical, beta-turn, and possibly "random" structures. The above conclusion is surprisingly close to the secondary structure of the "core" protein of pig skin proteodermatan sulfate proposed in the previous paper (V. Renugopalakrishnan et al., Biopolymers 28, 1923-1933, 1989) from FT-IR and CD spectroscopic studies in aqueous solution.     

Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression

The secondary structures of proteins (alpha-helical, beta-sheet, beta-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r(2) values of 0.9974, 0.9864, 0.9924, and 0.9743 for alpha-helical, beta-sheet, beta-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r(2) values (r(2) are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 895-905, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com