Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

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.     

Triple helical structure and stabilization of collagen-like molecules with 4(R)-hydroxyproline in the Xaa position

In this study, we examine the relationships between the structure and stability of five related collagen-like molecules that have hydroxyproline residues occupying positions not observed in vertebrate collagen. Two of the molecules contain valine or threonine and form stable triple helices in water. Three of the molecules contain allo-threonine (an enantiomer of threonine), serine, or alanine, and are not stable. Using molecular dynamics simulation methods, we examine possible explanations for the stability difference, including considering the possibility that differences in solvent shielding of the essential interchain hydrogen bonds may result in differences in stability. By comparing the structures of threonine- and allo-threonine-containing molecules in six polar and nonpolar solvation conditions, we find that solvent shielding is not an adequate explanation for the stability difference. A closer examination of the peptides shows that the structures of the unstable molecules are looser, having weaker intermolecular hydrogen bonds. The weakened hydrogen bonds result from extended Yaa residue Psi-angles that prevent optimal geometry. The Phi-Psi-maps of the relevant residues suggest that each residue's most favorable Psi-angle determines the corresponding collagen-like molecule's stability. Additionally, we propose that these molecules illustrate a more general feature of triple-helical structures: interchain hydrogen bonds are always longer and weaker than ideal, so they are sensitive to relatively small changes in molecular structure. This sensitivity to small changes may explain why large stability differences often result from seemingly small changes in residue sequence.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

The crystal structure of SDR-type pyridoxal 4-dehydrogenase of Mesorhizobium loti

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B(6) compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD(+)-bound enzyme.     

The crystal structure of a collagen-like polypeptide with 3(S)-hydroxyproline residues in the Xaa position forms a standard 7/2 collagen triple helix

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)-hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 A resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cgamma-endo), and the varphi/psi dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues. Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix.     

X-ray studies of water structure in 2 Zn insulin crystal

The water structure of rhombohedral 2 Zn insulin crystal which contains about 280 water molecules and 0.55-0.60 mol citrate molecules per dimer has been studied by X-ray crystallographic refinement with 1.1 A resolution data. Atomic parameters of 83 fully occupied and 258 partially occupied water molecules and 0.3 mol of citrate were obtained. Full matrix least-squares method with isotropic temperature factor was used for the refinement of partially occupied water molecules. The water molecules in this crystal exist in one of the three states: fully occupied water, partially occupied water and water continuum, and a schematic model of water structure in protein crystal was proposed. The flexibility of water molecules is described.     

The crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding

Lipocalins form a large multifunctional family of small proteins (15-25 kDa) first discovered in eukaryotes. More recently, several types of bacterial lipocalins have been reported, among which Blc from Escherichia coli is an outer membrane lipoprotein. As part of our structural genomics effort on proteins from E. coli, we have expressed, crystallized and solved the structure of Blc at 1.8 A resolution using remote SAD with xenon. The structure of Blc, the first of a bacterial lipocalin, exhibits a classical fold formed by a beta-barrel and a alpha-helix similar to that of the moth bilin binding protein. Its empty and open cavity, however, is too narrow to accommodate bilin, while the alkyl chains of two fatty acids or of a phospholipid could be readily modeled inside the cavity. Blc was reported to be expressed under stress conditions such as starvation or high osmolarity, during which the cell envelope suffers and requires maintenance. These data, together with our structural interpretation, suggest a role for Blc in storage or transport of lipids necessary for membrane repair or maintenance.     

Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.     

Sequence position of 3-hydroxyproline in basement membrane collagen. Isolation of glycyl-3-hydroxyprolyl-4-hydroxyproline from swine kidney.

The position of 3-hydroxyproline was investigated in the triplet sequences of peptides released by collagenase digestion of a collagen preparation from kidney cortex. Composition of the collagen preparation indicated that it was largely or wholly of basement membrane origin. 3-Hydroxyproline was detected in only one sequence, the tripeptide, glycyl-3-hydroxyprolyl-4-hydroxyproline, which accounted for a major fraction of the total 3-hydroxyproline obtained in the peptides released by collagenase. Preliminary data, based on sequencing the peptide mixture released by collagenase treatment, suggested that, in contrast, 4-hydroxyproline occurs predominantly if not exclusively in the Y position of Gly-X-Y triplet sequences in the collagen preparation studied.     

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.     

Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

The crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

The crystal structure of L-chiro-inositol

The crystal structure of L-chiro-inositol is monoclinic, P21, with a = 6.867(3), b = 9.133(4), c = 6.217(3) A, beta = 106.59(4) degrees, Z = 2. The structure was solved by using MULTAN, and refined to R = 0.028 for 1065 intensities observed with Ni-filtered MoK alpha radiation. The molecule has the expected chair conformation, with puckering parameters Q = 0.561 A, theta = 4.4 degrees, phi = 51.2 degrees. The non-hydrogen molecular symmetry is close to C2, with deviations of less than 0.07 A from a weighted fit. The intramolecular hydrogen-bonding forms infinite chains which are cross-linked through the weaker component of a three-center bond. The C-C bond lengths range from 1.515 to 1.528 A, and the C-O bond lengths from 1.418 to 1.436 A. The C-C-C angles range from 109.7 to 113.1 degrees, and the C-C-O angles from 106.5 to 112.0 degrees.