Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

The uniqueness of protein sequences: A Monte Carlo analysis

Amino acid sequences have already been examined in some detail in order to relate them to structural aspects, homology and gene duplication. This report introduces the concept of internal uniqueness of tripeptides within protein sequences and uses the Monte Carlo method to study this property. Some idea of internal uniqueness may be obtained from such an analysis using only a single sequence if the probability of the random occurrence is about 0.001 or less. This method of analysis is similar to that used in quantitative evaluations of homology. When the probability of the random occurrence is larger than 0.001 a homologous group of sequences is required and the random probabilities may be compared with the real occurrences within the group. From such an examination insulin and cytochrome c are identified as protein sequences with high internal uniqueness. A comparison of data from internal uniqueness and gene duplication analyses shows that these two properties need not be related. Results of the analysis point to internal uniqueness as an additional parameter for inclusion in speculations on why twenty amino acids are coded in protein structure.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

Region-specific role of water in collagen unwinding and assembly

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (PDB ID: 1BKV). A torsional map approach was used to characterize the conformational motion of the molecule that differ between imino-rich and imino-poor regions. At temperatures 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. We found that unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 A, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly.     

Deciphering the mechanisms of intestinal imino (and amino) acid transport: The redemption of SLC36A1

The absorption of zwitterionic imino and amino acids, and related drugs, is an essential function of the small intestinal epithelium. This review focuses on the physiological roles of transporters recently identified at the molecular level, in particular SLC36A1, by identifying how they relate to the classical epithelial imino and amino acid transporters characterised in mammalian small intestine in the 1960s-1990s. SLC36A1 transports a number of d- and l-imino and amino acids, β- and γ-amino acids and orally-active neuromodulatory and antibacterial agents. SLC36A1 (or PAT1) functions as a proton-coupled imino and amino acid symporter in cooperation with the Na⁺/H⁺ exchanger NHE3 (SLC9A3) to produce the imino acid carrier identified in rat small intestine in the 1960s but subsequently ignored because of confusion with the IMINO transporter. However, it is the sodium/imino and amino acid cotransporter SLC6A20 which corresponds to the betaine carrier (identified in hamster, 1960s) and IMINO transporter (identified in rabbit and guinea pig, 1980s). This review summarises evidence for expression of SLC36A1 and SLC6A20 in human small intestine, highlights the differences in functional characteristics of the imino acid carrier and IMINO transporter, and explains the confusion surrounding these two distinct transport systems.     

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 μm silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 × 10³ M⁻¹ s⁻¹ with solid phase rate constants for the same sequences measured at 2.5 × 10² M⁻¹ s⁻¹. The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.     

Thermodynamic stabilities of internal loops with GU closing pairs in RNA

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.     

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base-pair sequence 5′G-T-A-T-C-C-C-T-C-T-G-G-A-T-A-C3′ linked on the ends by dodecyl chains

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C¹²H²⁴ (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T₄ loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na⁺ and revealed, regardless of [Na ⁺], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T₄ loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T₄ end loops are essentially two state in 25 and 55 mM Na⁺. In contrast, significant deviations from two-state behavior were observed in 85 m MNa⁺. From our previously published melting data of DNA dumbbells with T_n end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881–892] and the dumbbell with T₄ end loops of this study, a plot of d(T_m)/d ln [Na⁺] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (T_m)/d ln [Na⁺] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.     

Factors affecting thermodynamic stabilities of RNA 3 x 3 internal loops

Internal loops in RNA are important for folding and function. The 3 x 3 nucleotide internal loops are the smallest size symmetric loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodynamic and structural insights acquired for 3 x 3 loops should improve approximations for stabilities of 3 x 3 and larger internal loops. Most natural 3 x 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides containing different 3 x 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodynamic stabilities of 3 x 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of approximately 1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 x 3 loops is derived by multiple linear regression.     

The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [¹⁴C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or α-α-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.     

Comprehensive conformational analysis of (Gly-Pro-Pro)n and (Gly-Pro-Hyp)n related to collagen

A complete analysis of all possible conformations with correct hydrogen bonds of the collagen II type was performed on the basis of developed simultaneous equations. Using a unimodal search (by varying Ψ³), the energetically favorable structure was obtained. No other energetically satisfactory structural solutions are possible. The next aim was to obtain a precise model of the molecule. The program used includes a subroutine for continual deformation of the pyrrolidine rings. The set of parameters determining the structure consists of 14 independent variables (8 dihedral and 6 bond angles). As starting points for the energy optimization, conformations produced by scanning and some structures from previous work were used. The final structures (practically the same for both polymers) have helix parameters h = 0.285 nm and t = 52°, which are in excellent agreement with the 7/2 symmetry of diffraction data. The conformations of the pyrrolidine rings are of the B type, i.e., C₂-C^β-exo-C^γ-endo. For both polypeptides, the conformations of imino acids in position 3 of the triplet are the same; in position 2, however, they are slightly different. The difference in diffraction patterns for the 7/2 and 10/3 helices is discussed.     

Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ～ 1650 cm^?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm^?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm^?1 component in the native collagen spectrum diminishes and the 1633-cm^?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm^?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.     

The distribution of oocyte 5S,somatic 5S and 18S + 28S rDNA sequences in the lampbrush chromosomes of Xenopus laevis

The nucleolus organizer locus of Xenopus laevis lampbrush chromosomes was identified by in situ hybridization of a ³H-labelled probe complementary to 18S + 28S rDNA. The nucleolus organizer is an axial granule on chromosome III that lies four-fifths the way down this chromosome reading from its larger (left) telomere, just within an exploded region that extends to its right end, where the lateral loops are exceptionally long. By in situ hybridization of ³H-labelled oocyte and somatic 5S spacer cRNA probes to similarly RNase-treated and denatured lampbrush chromosomes, the multiple sites of oocyte and somatic 5S gene families were identified. Oocyte 5S genes lie at the larger telomeres of the 15 chromosomes that possess these structures; that is, all but chromosomes X, XVII and XVIII. There are a further four sites, all peripheral, and in three of these, on chromosomes VII, X and XI, the sequences lie on lateral loops that are resolvable with the light microscope. By contrast all of the somatic 5S gene clusters occupy peripheral sites. There are two sites on chromosome III, one of which may be shared with oocyte 5S sequences; one on chromosome VII, which is very likely shared with oocyte 5S sequences; one terminal site on chromosome X; one site on chromosome XI that lies on a single pair of long loops which are inserted in a conspicuous and recognizable axial granule, loops which certainly carry oocyte 5S sequences too; two nearly terminal sites alongside the larger telomeres on chromosomes XII and XIV; and single interstitial sites on all three of the sphere-bearing chromosomes, VIII, IX and XVI. We suggest that 5S sequences on resolvable loops are transcribed by readthrough from upstream promoters, probably by polymerase II.     

Why do protein architectures have boltzmann-like statistics?

A theoretical study has shown that the occurrence of various structural elements in stable folds of random copolymers is exponentially dependent on the own energy of the element. A similar occurrence-on-energy dependence is observed in globular proteins¹ from the level of amino acid conformations to the level of overall architectures. Thus, the structural features stabilized by many random sequences are typical of globular proteins while the features rarely observed in proteins are those which are stabilized by only a minor part of the random sequences. © 1995 Wiley-Liss, Inc.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the mechanisms of the influence of imino acids on the physical characteristics of collagens

The influence of imino acids on the thermodynamic characteristics of collagen type structures in various collagens has been analyzed. It was shown that the basic mechanism of entropy increase in the protein-water system consists in the alteration in the number of cooperative segments accompanying the increase in imino acids content, which can be observed during the melting of the fibrous macromolecule. The range of variation in the physical characteristics of cooperative units is determined, in particular, by the variability of hydrogen bond parameters. This is displayed in a broadening of the bands of NH-valence vibrations and the half-widths of transitions in post-denatured structures. Thus, the basic mechanism of the influence of imino acids on thermodynamic characteristics of collagens is related to the complex nature of the melting process. The dehydration-hydration mechanisms of native and denatured states become significantly different upon replacement of any amino acid by an imino acid.     

Far-infrared spectroscopy of salt penetration into a collagen fiber scaffold

We employed far-infrared spectroscopy to observe the amount of salt that penetrates into collagen fiber masses. The absorption properties of collagen sheets prepared from tilapia skin, bovine skin, rat tail, and sea cucumber dermis were measured using a transmission Fourier transform spectrometer in a band from approximately 100 to 700 cm⁻¹. We confirmed that the absorbance spectra of the four types of dried collagen sheet show good agreement, even though the amino acid compositions differed. The absorbance peaks observed in the band corresponded to collective vibrations of plural functional groups such as methylene and imino groups in collagen. When salt solution was added to the collagen sheets and then dried, the spectral shapes of the sheets at approximately 166 cm⁻¹ were clearly different from those of the plain collagen sheets. The differential absorbance between wavenumbers 166 cm⁻¹ and 250 cm⁻¹ sensitively reflected the difference between higher-order structures, and the salt diffusion (crystallization) depended on the collagen fiber condition. From these results, we consider that spectral changes can be used for the numerical evaluation of salt penetration into a collagen fiber scaffold.     

Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, Clq and acetylcholinesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the β-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific β-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to Clq and acetylcholineesterase both of which share the general structural and functional properties of collagen in their “tail” regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of Clq and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid

Bacteriophage R17 RNA was labelled with ³²P and was subjected to partial digestion with ribonuclease T₁. The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T₁. Fragment 20 had a sequence that coded for the amino acids in positions 32–53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56–66 and 67–76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.