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.     

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.     

Stability and mobility of the collagen structure.

The thermodynamic analysis of microcalorimetric and hydrogen-exchange data on the stability and mobility of collagen structures from different species with different physiological temperatures has shown that not only the thermostability but the enthalpy and entropy of disruption of the native collagen structure are increasing functions of the total prolyl and hydroxyprolyl content. At the same time the number of stable hydrogen bonds maintaining the native structure is constant and consists of one stable and a less stable (0.7) bond per triplet for all the collagens studied, in agreement with Ramachandran's model (Ramachandran &; Kartha, 1955). Thus the observed difference between the enthalpies of disruption of collagens with a different imino acid content cannot be explained by the difference in the amount of stable hydrogen bonds involved in maintaining the native collagen structure. With an increase in the imino acid content of collagen, the Gibbs energy of micro-unfolding, which determines the mobility of a structure, also increases. It seems probable that the rigidity and order of the core of a native macromolecule influence the order of the surrounding water and that they are the main source of the observed large values for the enthalpy and entropy of collagen unfolding. The observed link between the stability and mobility properties of collagens explains the correlation that exists between the thermostabilities of collagens and the physiological temperatures of different species if it is assumed that some definite level of mobility of collagen structure is required for its efficient functioning in living systems. The experimental data presented here support the validity of this assumption.     

A study of the biosynthesis of collagenous and non-collagenous proteins and of the proline/hydroxyproline ratios of collagens isolated from embryonic tissues

1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.     

The triple helix-coil transition of cyanogen-bromide peptides of the α1-chain of the calf-skin collagen

The thermal triple helix-coil transition of the CNBr peptides of the α1-chain of calf-skin collagen was studied optically and calorimetrically. Besides α1CB5, all the peptides were able to form triple-helical structures at low temperatures. The peptides with longer chain lengths showed, under the experimental conditions, hysteresis in the transition range depending on the direction of the successive temperature changes. The detailed thermodynamic analysis of the optical transition curves was only possible for the two small peptides α1CB2 and α1CB4. We observed a higher stability of α1CB2 relative to α1CB4 (α1CB2 has higher imino acid content), accompanied with increased values of both denaturation enthalpy and entropy. Further, we observed a linear relationship between the calorimetrically determined denaturation enthalpy of all the CNBr peptides and their imino acid content. Although this behavior is qualitatively in accordance with the observation of Privalov and Tiktopulo on various kinds of native collagen, the CNBr peptides showed much lower values of the thermodynamic parameters ΔH⁰ and ΔS⁰ and differed also in the rate of their change with imino acid content. These differences are interpreted as being caused by misalignment in the helical form of the CNBr peptides resulting in a rupture of the specific interactions in the native form.     

A model for interstitial collagen catabolism by mammalian collagenases.

Mammalian collagenases cleave all three alpha chains of native, triple-helical types I, II, and III collagens after the Gly residue of the partial sequence Gly-[Ile or Leu]-[Ala or Leu] at a single locus approximately three-fourths from the amino terminus. There are an additional 31 sites in the triple-helical regions of types I, II, III, and IV collagens that contain the same partial sequence but are not hydrolyzed. A model has been developed to explain this remarkable specificity. The mammalian collagenase cleavage site in interstitial collagens is distinguished by: (a) a low side-chain molal volume-, high imino acid (greater than 33%)-containing region that is tightly triple-helical, consisting of four Gly-X-Y triplets preceding the cleavage site, (b) a low imino acid-containing (less than 17%), loosely triple-helical region consisting of four Gly-X-Y triplets following the cleavage site, and (c) a maximum of one charged residue for the entire 25 residue cleavage site region, which is always an Arg that follows the cleavage site in subsite P'5 or P'8. In addition, the high imino acid-containing region cannot have an imino acid adjacent to the cleaved Gly-[Ile or Leu] bond (i.e. in subsite P2). Careful scrutiny of the 31 non-cleaved sequences reveals that none of those sites shares all of the characteristics of the cleavage site. The criterion of this model thus explain both cleaved and non-cleaved sequences in the triple-helical regions of types I, II, III, and IV collagen, and are supported by all known experimental and theoretical results on collagen catabolism and structure.     

The number of cooperative regions (energetic domains) in a pepsin molecule depends on the pH of the medium

The technique of scanning microcalorimetry was used to study the effect exerted by ethanol and by the pH of the medium on the number and size of cooperative regions in a pepsin molecule. Ethanol addition lowered the temperature of protein denaturation, but did not change the number of energetic domains. The number of thermodynamic cooperative units (determined as a delta Hcal to delta Heff ratio) was reduced from four to two when the pH changed from 6.7 to 2.0. As was demonstrated using the CD technique, this process involved no changes either in the secondary structure or in the local surroundings of aromatic amino acids. Therefore, variations in the cooperative properties of a pepsin globule at different pH values are associated with the electrostatic interactions of individual parts of the molecule.     

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas

Type I collagens were extracted from fish scales of Pagrus major and Oreochromis niloticas as a possible underutilized resource for medical materials. The fish scales were demineralized with EDTA and digested by pepsin. The resultant type I collagens contained more than 33.6% of glycine as the most abundant amino acid. The denaturation temperatures of the collagens from P. major and O. niloticas were 303 and 308 K, respectively, both of which were relatively lower than that of porcine dermis collagen (314 K). CD spectra indicated that the denaturation temperatures were dependent on the amount of hydroxyproline, rather than proline residues. Raman spectra also indicated that the relative intensities of Raman lines at 879 and 855 cm⁻¹ assigned to Hyp and Pro rings were changed due to the contents of the imino acids. Significantly, the content of sulphur-containing methionine was higher in the fish scales than in porcine dermis. The enthalpy and entropy estimated from thermal analyses could be correlated to amino acid sequences (Gly-Pro-Hyp) of type I collagens and the number of methionine amino acid residues.     

A study of the hydration and thermodynamics of warm-water and cold-water fish collagens.

The hydrated volumes, Vh, of collagens extracted from various fish species were calculated by using the Simha-Einstein equation, and it was found that the hydration of warm-water fish collagen is greater than that of cold-water fish collagen (halibut). Although the intrinsic viscosities of warm-water fish (bigeye-tuna, carp and catfish) collagens are almost the same, the hydrated volume of bigeye-tuna collagen is approx. 1.5 and 3 times those of carp and catfish collagens respectively. The extent of hydration at 20 degrees C is in the following order: bigeye tuna greater than carp greater than catfish greater than halibut. The various thermodynamic activation parameters (delta G*, delta H* and delta S*) were calculated and it was found that they are useful for determining the exact denaturation temperature. It was calculated that the denaturation temperatures of halibut, bigeye-tuna, carp and catfish collagens are 17, 31, 32 and 26-30 degrees C respectively. The variations of hydration, intrinsic viscosity, denaturation temperature and the thermodynamic parameters with the variation of concentration of catfish collagen were also thoroughly examined. The change of thermodynamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results.     

New analysis of the phylogenetic change of collagen thermostability

Recent data concerning the thermostability and the primary structure of type IV collagens, some invertebrate collagens, and for the stability of synthetic collagen-like polypeptides, show that our earlier analysis of the phylogenetic change of thermostability has some shortcomings. The results of the analysis were corrected and it has been shown that the dependence of denaturation temperature Td on 4-hydroxyproline content is hyperbolic and the total Gly-Pro-Hyp sequence content is a main, but not exclusive, factor influencing the change of collagen thermostability. It appears possible that the same mechanism underlies the thermostability of fibril-forming collagens of all animal life, ranging from Antarctic ice fish to at least one annelid (Alvinella pompejana) living at very high temperatures at the bottom of the ocean near thermal vents.     

Number of cooperative regions (energy domains) in the pepsin molecule depends on the pH of the medium]

Ethanol and pH influence on the number and dimensions of cooperative regions in pepsin molecule was studied by scanning microcalorimetry. It is shown that ethanol solution causes a decrease of temperature of protein denaturation but does not influence the number of energetic domains. While changing pH from 6.7 to 2.0 the number of thermodynamic cooperative units (defined as the ratio delta Hcal/delta Heff) decreases from four to two. This process, as shown by CD technique, is followed by changes neither in the secondary structure, nor in the local environment of aromatic amino acids. A conclusion is made that the distinctions in cooperative characteristics of the protein globule at different pH are determined by electrostatic interactions of separate parts of the molecule.     

Reactivity of the imino acids formed in the amino acid oxidase reaction.

The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.     

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, beta- and gamma-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.     

Structure, dynamics, and thermodynamics of mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

Stabilization of collagen structure: Dependence of collagen denaturation enthalpy on the imino acid content

An analysis of the available data on the enthalpy (ΔH_r) of denaturation (melting) of collagens with different imino acid content in solution and in the aggregated state has shown that ΔH_r in solution increases with increasing denaturation temperature, whereas in the aggregated state there is an inverse dependence. ΔH_r in solution correlates with the hydroxyproline content but not with that of proline. No correlation between the change of ΔH_r and the imino acid content is observed for the aggregated state.     

Can G-C Hoogsteen-wobble pairs contribute to the stability of d(G. C-C) triplexes?

Quantum mechanics, molecular dynamics and statistical mechanics methods are used to analyze the importance of neutral Hoogsteen-wobble G.C pairing in the stabilization of triple helices based on the poly-(G.C-C) trio at neutral pH and low ionic strength. In spite of the existence of a single hydrogen bond, the Hoogsteen-wobble G.C pair is found to be quite stable both in gas phase and solvated DNA. Molecular dynamics simulations of different triplexes based on the d(G.C-C) trio leads to stable structures if the neutral d(G.C-C) steps stabilized by Hoogsteen-wobble pairs are mixed with d(G.C-C+) steps. Finally, high level ab initio calculations and thermodynamic integration techniques are used to determine the relative stability of G.C wobble and G.C imino pairings. It is found that triplexes containing the imino pairing are slightly more stable structures than those with the wobble one, due mainly to a better stacking.     

Influence of growth temperature on the structure and thermodynamic parameters of barley starches

Barley starches grown at different temperatures were investigated using high sensitivity differential scanning microcalorimetry and X-ray diffraction. By applying physico-chemical approaches, thickness of crystalline lamellae, thermodynamic and structural characteristics (such as gelatinisation) of cooperative units and parameters characterising thermodynamic properties of crystal surfaces were determined. It was established that a difference of growth temperature experienced by plants during development does not lead to changes in the thickness of amylopectin crystalline lamellae and hence constituent double helix length. The role of defects in structural organisation of native barley starches is discussed. It is suggested that not all fatty acids necessarily form crystalline inclusion complexes.     

Collagen denaturation enthalpy--nonlinear function of 4-hydroxyproline

The results of calorimetric measurements of denaturation of collagens with different imino acid content are reported. In contrast to the existing point of view that denaturation enthalpy is a linear function of 4-oxyproline content, a nonlinear dependence was revealed. It is suggested that the reason for the observed nonlinearity is triplets of the (Gly-Pro-Hyp) type. An increase of their content can cause a decrease in the denaturation enthalpy in accord with the water-bridge structure and due to the minimum enthalpy effect of stabilization of the triplets as compared to triplets of other type.