No abstract

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.

[structure: see text] Collagen is the most abundant protein in animals. Interstrand N-H...O=C hydrogen bonds between backbone amide groups form a ladder in the middle of the collagen triple helix. Isosteric replacement of the hydrogen-bond-donating amide with an ester or (E)-alkene markedly decreases the conformational stability of the triple helix. Thus, this recurring hydrogen bond is critical to the structural integrity of collagen. In this context, an ester isostere confers more stability than does an (E)-alkene.

Among the proteinogenic amino acids, only proline is a secondary amine and only proline has a saturated ring. Electronegative substituents on C-4 (that is, C(gamma)) have a substantial effect on the trans/cis ratio of the prolyl peptide bond and the pucker of the pyrrolidine ring. 2-Azabicyclo[2.1.1]hexane is, in essence, a proline analogue with two C(gamma) atoms, one in each of the two prevalent ring puckers of proline. Here, 2-azabicyclo[2.1.1]hexane analogues of 2S-proline, (2S,4S)-4-hydroxyproline, and (2S,4S)-4-fluoroproline residues were synthesized, and their trans/cis ratios were shown to be invariant in a particular solvent. Thus, the substitution of a proline residue on C-4 affects the trans/cis ratio by altering the pucker of its pyrrolidine ring. This finding has implications for the conformation of collagen, which has an abundance of 2S-proline and (2S,4R)-4-hydroxyproline residues, and can be stabilized by (2S,4R)-4-fluoroproline and (2S,4S)-4-fluoroproline residues.

Homing endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with extremely high specificity. I-PpoI endonuclease, a homing endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) of known three-dimensional structure that catalyzes the cleavage of a long target DNA sequence (15 base pairs). Here, a detailed chemical mechanism for catalysis of DNA cleavage by I-PpoI endonuclease is proposed and tested by creating six variants in which active-site residues are replaced with alanine. The side chains of three residues (Arg61, His98, and Asn119) are found to be important for efficient catalysis of DNA cleavage. This finding is consistent with the proposed mechanism in which His98 abstracts a proton from an attacking water molecule bound by an adjacent phosphoryl oxygen, Arg61 and Asn119 stabilize the pentavalent transition state, and Asn119 also binds to the essential divalent metal cation (e.g., Mg(2+) ion), which interacts with the 3'-oxygen leaving group. Because Mg(2+) is required for cleavage of a substrate with a good leaving group (p-nitrophenolate), Mg(2+) likely stabilizes the pentavalent transition state. The pH-dependence of k(cat) for catalysis by I-PpoI reveals a macroscopic pK(a) of 8.4 for titratable groups that modulate product release. I-PpoI appears to be unique among known restriction endonucleases and homing endonucleases in its use of a histidine residue to activate the attacking water molecule for in-line displacement of the 3'-leaving group.

In collagen, strands of the sequence XaaYaaGly form a triple‐helical structure. The Yaa residue is often (2S,4R)‐4‐hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O‐acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cγ atom: AcHyp[C(O)CF3]OMe ≅ Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe ≅ Achyp[C(O)CH3]OMe ≥ AcHypOMe ≅ AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)10, which is O‐acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)10 > {ProHyp[C(O)CH3]Gly}10 ≫ (ProProGly)10. Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

2′‐Fluoro‐2′‐deoxyuridine 3′‐phosphate (dUFMP) and arabinouridine 3′‐phosphate (araUMP) have non‐natural furanose rings. dUFMP and araUMP were prepared by chemical synthesis and found to have three‐ to sevenfold higher affinity than uridine 3′‐phosphate (3′‐UMP) or 2′‐deoxyuridine 3′‐phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3′‐UMP, dUFMP, and araUMP (pKa = 5.9) being more anionic than that of dUMP (pKa = 6.3). The three‐dimensional structures of the crystalline complexes of RNase A with dUMP, dUFMP and araUMP were determined at < 1.7 Å resolution by X‐ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3′‐UMP and dUFMP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A·araUMP complex, the 2′‐hydroxyl group is exposed to the solvent. All four 3′‐nucleotides bind more tightly to wild‐type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3′‐nucleotides, and indicate that non‐natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.

Page 2062. "(R)-MTPA-Cl" should be changed to "(S)-MTPA-Cl" (18 lines from the bottom of column two). This is correctly stated 28 lines from the top of the same column.Page 2062. "IPEA" should be changed to the more conventional "DIEA" eight lines from the bottom of column two.Page 2063. The header "(6R)-2,6-Dimethyl-1,4,5,6-tetrahydropyridine-(R)-MTPA Amide (12) and -(S)-MTPA Amide (13)" should be changed to "(6R)-and (6S)-2,6-Dimethyl-1,4,5,6-tetrahydropyridine-(R)-MTPA Amides (12 and 13, Respectively)" in column two.