On the stability of different experimental dimeric structures of the SL1 sequence from the genomic RNA of HIV-1 in solution: a molecular dynamics simulation and electrophoresis study

SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.     

Molecular dynamics simulations of Trp side-chain conformational flexibility in the gramicidin A channel

Gramicidin A (gA) is prototypical peptide antibiotic and a model ion channel former. Configured in the solid-state NMR beta(6.5)-helix channel conformation, gA was subjected to 1-ns molecular dynamics (MD) gas phase simulations using the all-atom charmm22 force field to ascertain the conformational stability of the Trp side chains as governed by backbone and neighboring side-chain contacts. Three microcanonical trajectories were computed using different initial atomic velocities for each of twenty different initial structures. For each set, one of the four Trp side chains in each monomer was initially positioned in one of the five non-native conformations (A. E. Dorigo et al., Biophysical Journal, 1999, Vol. 76, 1897-1908), the other Trps being positioned in the native state, o1. In three additional control simulations, all Trps were initiated in the native conformation. After equilibration, constraints were removed and subsequent conformational changes of the initially constrained Trp were measured. The chi(1) was more flexible than chi(2.1). The energetically optimal orientation, o1 (Dorigo et al., 1999), was the most stable in all four Trp positions (9, 11, 13, 15) and remained unchanged for the entire 1 ns simulation in 19 of 24 trials. Changes in chi(1) from each of the 5 suboptimal states occur readily. Two of the non-native conformations reverted readily to o1, whereas the other three converted to an intermediate state, i2. There were frequent interconversions between i2 and o1. We speculate that experimentally observed Trp stability is caused by interactions with the lipid-water interface, and that stabilization of one of the suboptimal conformations in gA, such as i2, by lipid headgroups could produce a secondary, metastable conformational state. This could explain recent experimental studies of differences in the channel conductance dispersity between gA and a Trp-to-Phe gA analog, gramicidin M (gM, J. C. Markham et al., Biochimica et Biophysica Acta, 2001, Vol. 1513, 185-192).     

Stretching short DNAs in electrolytes

This paper is aimed at a combined theoretical and numerical study of the force-extension relation of a short DNA molecule stretched in an electrolyte. A theoretical formula based on a recent discrete wormlike chain (WLC) model of Kierfeld et al. (Eur Phys. J. E, Vol. 14, pp.17-34, 2004) and the classical OSF mean-field theory on electrostatic stiffening of a charged polymer is numerically verified by a set of Brownian dynamics simulations based on a generalized bead-rod (GBR) model incorporating long-ranged electrostatic interactions via the Debye-Hueckel potential (DH). The analysis indicates that the stretching of a short DNA can be well described as a WLC with a constant effective persistent length. This contrasts the behavior of long DNA chains that are known to exhibit variable persistent lengths depending on the ion concentration levels and force magnitudes.     

Brownian dynamics simulations of glycolytic enzyme subsets with F-actin

Previous Brownian dynamics (BD) simulations identified specific basic residues on fructose-1,6-bisphophate aldolase (aldolase) (I. V. Ouporov et al., Biophysical Journal, 1999, Vol. 76, pp. 17-27) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I. V. Ouporov et al., Journal of Molecular Recognition, 2001, Vol. 14, pp. 29-41) involved in binding F-actin, and suggested that the quaternary structure of the enzymes may be important. Herein, BD simulations of F-actin binding by enzyme dimers or peptides matching particular sequences of the enzyme and the intact enzyme triose phosphate isomerase (TIM) are compared. BD confirms the experimental observation that TIM has little affinity for F-actin. For aldolase, the critical residues identified by BD are found in surface grooves, formed by subunits A/D and B/C, where they face like residues of the neighboring subunit enhancing their electrostatic potentials. BD simulations between F-actin and aldolase A/D dimers give results similar to the native tetramer. Aldolase A/B dimers form complexes involving residues that are buried in the native structure and are energetically weaker; these results support the importance of quaternary structure for aldolase. GAPDH, however, placed the critical residues on the corners of the tetramer so there is no enhancement of the electrostatic potential between the subunits. Simulations using GAPDH dimers composed of either S/H or G/H subunits show reduced binding energetics compared to the tetramer, but for both dimers, the sets of residues involved in binding are similar to those found for the native tetramer. BD simulations using either aldolase or GAPDH peptides that bind F-actin experimentally show complex formation. The GAPDH peptide bound to the same F-actin domain as did the intact tetramer; however, unlike the tetramer, the aldolase peptide lacked specificity for binding a single F-actin domain.     

NMR conformational analysis of proadrenomedullin N-terminal 20 peptide, a proangiogenic factor involved in tumor growth

The preferred conformation of Proadrenomedullin N-Terminal 20 Peptide (PAMP; ARLDVASEFRKKWNKWALSR-amide) has been determined using 1H and 13C two-dimensional nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. PAMP is a peptide that has various physiological functions, including its role as a proangiogenic factor in facilitating tumor growth and its inhibitory effect on catecholamine secretion at nicotinic receptors. The preferred conformation of PAMP was determined in a helix-inducing trifluoroethanol and water (TFE/H2O) solution, and in a membrane-mimetic sodium dodecylsulfate-d25 (SDS) micellar solution. The secondary structure consists of an alpha-helix for residues Arg2 to Arg20 in TFE/H2O solution and an alpha-helix for residues Arg2 to Ala17 in SDS solution. We postulate that the polar charged residues Arg2, Lys12, and Arg20 are responsible for the initial interaction of the peptide with the micelle, and that this is followed by the binding of the hydrophobic residues Leu3, Val5, Phe9, Trp13, and Trp16 to the micellar core. The three C-terminal amino acid residues adopt an extended structure in SDS, suggesting that they are important in receptor recognition and binding. This is supported by truncation studies done by Mahata et al. (Hypertension, 1998, Vol. 32, pp. 907-916), which show the importance of the C-terminal in physiological activity. Furthermore, Belloni et al. (Hypertension, 1999, Vol. 33, pp. 1185-1189), and Martinez et al. (Cancer Research, 2004, Vol. 64, pp. 6489-6494) suggested that the N-terminal was also important in PAMP activity. However, no differences in conformational preference of the N-terminal were observed between the two solvent systems.     

Molecular dynamics simulation studies of induced fit and conformational capture in U1A–RNA binding: Do molecular substates code for specificity?

Molecular dynamics (MD) simulations on stem loop 2 of U1 small nuclear RNA and a construct of the U1A protein were carried out to obtain predictions of the structures for the unbound forms in solution and to elucidate dynamical aspects of induced fit upon binding. A crystal structure of the complex between the U1A protein and stem loop 2 RNA and an NMR structure for the uncomplexed form of the U1A protein are available from Oubridge et al. (Nature, 1994, Vol. 372, pp. 432-438) and Avis et al. (Journal of Molecular Biology, 1996, Vol. 257, pp. 398-411), respectively. As a consequence, U1A-RNA binding is a particularly attractive case for investigations of induced fit in protein-nucleic acid complexation. When combined with the available structural data, the results from simulations indicate that structural adaptation of U1A protein and RNA define distinct mechanisms for induced fit. For the protein, the calculations indicate that induced fit upon binding involves a non-native thermodynamic substate in which the structure is preorganized for binding. In contrast, induced fit of the RNA involves a distortion of the native structure in solution to an unstable form. However, the RNA solution structures predicted from simulation show evidence that structures in which groups of bases are favorably oriented for binding the U1A protein are thermally accessible. These results, which quantify with computational modeling recent proposals on induced fit and conformational capture by Leuillot and Varani (Biochemistry, 2001, Vol. 40, pp. 7947-7956) and by Williamson (Nature Structural Biology, 2000, Vol. 7, pp. 834-837) suggest an important role for intrinsic molecular architecture and substates other than the native form in the specificity of protein-RNA interactions.     

Conformational study of endothelins and sarafotoxins with the cystine-stabilized helical motif by means of CD spectra.

A series of CD measurements were carried out on members of peptides in the endothelin and sarafotoxin families. The helical structures taken by these peptides containing the helical motif with the sequences of Cys-X-X-X-Cys and Cys-X-Cys [Y. Kobayashi et al. (1991) Neurochemistry International Vol. 18, pp. 525-534] are classified into three groups: a group of structures of ET-1, ET-2 and vasoactive intestinal contractor (VIC), a group of sarafotoxin, and a group of ET-3.     

The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.     

Conformational studies on a synthetic C-terminal fragment of the alpha subunit of G(S) proteins

It has recently been reported that synthetic peptides corresponding to the C-terminal sequence of G alpha, can be used to study the molecular mechanisms of interaction between this protein and G protein coupled receptors (Hamm et al., Science, 1988, Vol. 241, pp. 832-835). A conformational analysis on a 11 amino acids peptide from the G alpha(S) C-terminus, G alpha(S)(384-394) (H-QRMHLRQYELL-OH), was performed by nmr spectroscopy and molecular modeling methods. Two-dimensional nmr spectra, recorded in hexafluoroacetone/water, a mixture with structure stabilizing properties, showed an unusually high number of nuclear Overhauser effects, forming significative pattern to the drawing of a secondary structure. Conformations consistent with experimental NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers corresponding to an alpha-turn and a type III beta-turn involving the last five C-terminal residues. Interestingly, the alpha-turn conformation was found to overlap with good agreement the crystallographic structure of the same fragment in the G alpha(S) protein.     

Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state

The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.     

Spatial structure of myelopeptides: I. conformational analysis of MP-1, MP-2, and MP-3

Theoretical conformational analysis was used to study the spatial structure and conformational properties of myelopeptides, bone-marrow peptide mediators. The low-energy conformations of three hexapeptides MP-1 (Phe-Leu-Gly-Phe-Pro-Thr), MP-2 (Leu-Val-Val-Tyr-Pro-Trp), and MP-3 (Leu-Val-Cys-Tyr-Pro-Gln) were found, the values of dihedral angles of the backbone and side chains of the amino acid residues con-stituting these peptides were determined, and the energies of intra- and interresidual interactions were estimated.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 31–38.Original Russian Text Copyright © 2005 by Akhmedov, Ismailova, Abbasli, Akhmedov, Godjaev.     

Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase

A series of tripeptides that satisfy the -Asn-Xaa-Thr/Ser- primary sequence requirement [Marshall, R. D. (1972) Annu. Rev. Biochem. 41, 673-702] for N-glycosylation have been synthesized and examined as potential acceptors in an oligosaccharyltransferase assay. Of these, six (Ac-Asn-Ala-Thr-NH2, Ac-Asn-Leu-Thr-NH2, Ac-Asn-Asp-Thr-NH2, Ac-Asn-D-Ala-Thr-NH2, Ac-Asn-Pro-Thr-NH2, and Ac-Asn-AIB-Thr-NH2) were examined for solution conformational properties in dimethyl sulfoxide with use of amide proton temperature coefficients, 3JHN alpha analysis [Pardi, A., et al. (1984) J. Mol. Biol. 180, 741-751], and 2-D ROESY experiments [Bothner-By, A. A., et al. (1984) J. Am. Chem. Soc. 106, 811-813]. The analysis reveals that the peptides that serve as acceptors in the transferase assay demonstrate similar conformational properties in solution. These are highlighted by a secondary structural motif that involves the interaction between the asparagine side-chain carboxamide and the backbone amide of the threonine. The peptides that show very poor acceptor, or even nonacceptor, properties in the oligosaccharyltransferase assay demonstrate different conformational features in solution. These observations may explain the distinct biological activity observed for these peptides.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

Molecular dynamics simulations of small peptides: dependence on dielectric model and pH

There has been much interest recently in the structure of small peptides in solution. A recent study by Bradley and co-workers [(1989) in Techniques of Protein Chemistry, Hugli, T.E., Ed., Academic Press, Orlando, FL, pp. 531-546; (1990) Journal of Molecular Biology, 215, pp. 607-622] describes a 17-residue peptide that is stable as a monomeric helix in aqueous solution at low pH, as determined by two-dimensional nmr and CD spectroscopy. They also have determined the helix content of the peptide as a function of pH using CD. We performed molecular dynamics simulations, with an empirical force field, of this peptide at low pH, with three different dielectric models: a linear distance-dependent dielectric function (epsilon = R); a modified form [J. Ramstein and R. Lavery (1988) Proceedings of the National Academy of Science, USA, Vol. 85, pp. 7231-7235] of the sigmoidal distance-dependent dielectric function of Hingerty and co-workers [(1985) Biopolymers, Vol. 24, pp. 427-439]; and epsilon = 1 with the peptide immersed in a bath of water molecules. We found that simulations with the sigmoidal dielectric function and the model with explicit water molecules resulted in average distances for particular interactions that were consistent with the experimental nmr results, with the sigmoidal function best representing the data. However, these models exhibited very different helix-stabilizing interactions. We also performed simulations using the sigmoidal function at moderate and high pH to compare to experimental determinations of the pH dependence of helix content. Helix content did not decrease with increases in pH, as shown experimentally. We did, however, observe changes in a specific side chain-helix dipole interaction that was implicated in determining the pH-dependent behavior of this peptide. Overall, the sigmoidal dielectric function was a reasonable alternative to adding explicit water molecules. In comparing 100 ps molecular dynamics simulations, the sigmoidal function was much less computer intensive and sampled more of conformational space than the treatment using explicit water molecules. Sampling is especially important for this system since the peptide has been shown experimentally to populate both helical and nonhelical conformations.     

Comparison of conformational features of staphylococcal nuclease in ternary complexes with pdTp, pdGp, and nitrophenyl-pdTp.

The conformations of wild-type staphylococcal nuclease (SNase) in the ternary complexes with thymidine 3',5'-bisphosphate (pdTp), 2'-deoxyguanine 3',5'-bisphosphate (pdGp), and thymidine 3'-phosphate 5'-(p-nitrophenylphosphate) (NpdTp) with Ca2+ were examined by two-dimensional NMR NOESY and ROESY experiments. The results of these experiments indicate that the conformational features of the SNase are quite similar in the three ternary complexes. This suggests that the conformational features of SNase, in these ternary complexes, are not strongly dependent on whether the 5'-phosphate is a mono- or diester. This is in contrast to our prior studies on substitutions of active site charged amino acids which indicated that the conformational features of SNase in the ternary complex are quite sensitive to substitutions for active site charged amino acids (Hibler et al., 1987; Wilde et al., 1988; Pourmotabbed et al., 1990). The similarity of the SNase conformational features in the ternary complexes with pdTp and pdGp indicates that the features of the nucleotide bound at the active site are not strong determinants of the enzyme conformation in the ternary complexes. These conclusions are in general agreement with the results on pdApdT ternary complexes with SNase which suggested that it is the conformational features of the bound nucleic acid which determine the differences in catalysis observed for SNase with different substrates (Weber et al., 1991), more so than the conformational features of the enzyme.     

Immunocytochemical localization and biochemical analysis of alpha and beta keratins in the avian lingual epithelium

The alpha and beta keratins are found as 10-nm and 3-nm cytoplasmic filaments, respectively. While the alpha keratins are produced in essentially all vertebrate epithelia (Franke et al.: Exp. Cell Res., 116:429-445, 1978; Sun et al.: Proc. Natl. Acad. Sci. USA, 76:2813-2817, 1979), the beta keratins have been demonstrated only in specific epithelial tissues of birds and reptiles (Sawyer et al.: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 194-238, 1986; Landmann: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 150-187, 1986). Recently, Homberger and Brush (Zoomorphology, 106:103-114, 1986) have demonstrated that within the lingual epithelium of parrots, beta keratins are expressed exclusively in the anterior ventral region. While it is well established that epidermal-dermal interactions are important for the regional expression of the beta keratin genes in the avian scutate scales and feathers, little is known about the expression of beta keratins in other epithelial structures such as the tongue. We have used biochemical and immunocytochemical techniques to analyze the alpha and beta keratins of the lingual epithelium of the chick as an initial step in the characterization of this model system for developmental studies. We have found that alpha keratins are present throughout the lingual epithelium. The anterior ventral epithelium contains alpha keratin polypeptides characteristic of skin-type differentiation, while the epithelium of the dorsal and posterior ventral regions contains alpha keratin polypeptides characteristic of esophageal-type differentiation (O'Guin et al.: In: Current Topics in Developmental Biology: The Molecular and Developmental Biology of Keratins. A.A. Moscona and A. Monroy, eds. R.H. Sawyer, vol. ed. Academic Press, New York, Vol. 22, pp. 282-306, 1987). Beta keratins are produced only in the differentiated epithelial cells of the anterior ventral region of the tongue. Immunoelectron microscopy demonstrates that the alpha and beta keratins of the stratum intermedium and corneum of the anterior ventral region are found together in the large filament bundles characteristic of this region. The preexistence of the alpha keratins in the cells destined to produce beta keratins as well as the colocalization of these keratins in the filament bundles of these cells suggests that a functional relationship may exist between the alpha and beta keratins.     

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.     

Side-chain conformational entropy in protein unfolded states

The largest force disfavoring the folding of a protein is the loss of conformational entropy. A large contribution to this entropy loss is due to the side-chains, which are restricted, although not immobilized, in the folded protein. In order to accurately estimate the loss of side-chain conformational entropy that occurs upon folding it is necessary to have accurate estimates of the amount of entropy possessed by side-chains in the ensemble of unfolded states. A new scale of side-chain conformational entropies is presented here. This scale was derived from Monte Carlo computer simulations of small peptide models. It is demonstrated that the entropies are independent of host peptide length. This new scale has the advantage over previous scales of being more precise with low standard errors. Better estimates are obtained for long (e.g., Arg and Lys) and rare (e.g., Trp and Met) side-chains. Excellent agreement with previous side-chain entropy scales is achieved, indicating that further advancements in accuracy are likely to be small at best. Strikingly, longer side-chains are found to possess a smaller fraction of the theoretical maximum entropy available than short side-chains. This indicates that rotations about torsions after chi(2) are significantly affected by side-chain interactions with the polypeptide backbone. This finding invalidates previous assumptions about side-chain-backbone interactions. Proteins 2000;40:443-450.