Stabilization of short collagen-like triple helices by protein engineering.

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.

Glycosylation/Hydroxylation-induced stabilization of the collagen triple helix. 4-trans-hydroxyproline in the Xaa position can stabilize the triple helix.

We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr)(10)-NH(2) with beta-d-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr)(10)-NH(2) peptide, if the proline (Pro) in the Xaa position is replaced with 4-trans-hydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence Ac-(Gly-Pro-Thr(beta-d-Gal))(10)-NH(2) increases the T(m) of the triple helix by 15.7 degrees C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) form stable triple helices but the peptide (Hyp-Pro-Gly)(10) does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly)(10). Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr)(10)-NH(2) but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/vestimentiferan cuticle collagens.

Sweet is stable: glycosylation stabilizes collagen.

For most collagens, the melting temperature (T(m)) of the triple-helical structure of collagen correlates with the total content of proline (Pro) and 4-trans-hydroxyproline (Hyp) in the Xaa and Yaa positions of the -Gly-Xaa-Yaa- triplet repeat. The cuticle collagen of the deep-sea hydrothermal vent worm Riftia pachyptila, despite a very low content of Pro and Hyp, has a relatively high thermal stability. Rather than Hyp occupying the Yaa position, as is normally found in mammalian collagens, this position is occupied by threonine (Thr) which is O-glycosylated. We compare the triple-helix forming propensities in water of two model peptides, Ac-(Gly-Pro-Thr)(10)-NH(2) and Ac-(Gly-Pro-Thr(Galbeta))(10)-NH(2), and show that a collagen triple-helix structure is only achieved after glycosylation of Thr. Thus, we show for the first time that glycosylation is required for the formation of a stable tertiary structure and that this modification represents an alternative way of stabilizing the collagen triple-helix that is independent of the presence of Hyp.

Chicken FK506-binding protein, FKBP65, a member of the FKBP family of peptidylprolyl cis-trans isomerases, is only partially inhibited by FK506.

The chicken FK506-binding protein FKBP65, a peptidylprolyl cis-trans isomerase, is a rough endoplasmic reticulum protein that contains four domains homologous to FKBP13, another rough endoplasmic reticulum PPIase. Analytical ultracentrifugation suggests that in FKBP65 these four domains are arranged in a linear extended structure with a length of about 26 nm and a diameter of about 3 nm. All four domains are therefore expected to be accessible to substrates. The specificity of FKBP65 towards a number of peptide substrates was determined. The specific activity of FKBP65 is generally lower than that of FKBP12 when expressed as a per domain activity. The substrate specificity of FKBP65 also differs from that of FKBP12. Inhibition studies show that only one of the four domains can be inhibited by FK506, a powerful inhibitor of all other known FKBPs. Furthermore, the same domain seems to be susceptible to inhibition by cyclosporin A. No other FKBPs were shown to be inhibited by cyclosporin A. It is also shown that FKBP65 can catalyse the re-folding of type III collagen in vitro with a kcat/Km = 4.3 x 10(3) M-1.s-1.

Mutational analysis of the autoinhibitory domain of calmodulin kinase II.

Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

Protein dynamics and 1/f noise.

It has long been recognized that protein dynamical processes occur over a wide temporal range. However, the functionality of this spectrum of events remains unclear. In this work, a generalized noise function analysis is applied to a collection of diverse protein dynamical systems. It is shown that a power law model with an oscillatory component can adequately describe the time course of a variety of processes. These results suggest that under the appropriate conditions, proteins are in a metastable state. A microscopic, chemical kinetic model based on a Poisson distribution of activation energies is presented. From this model specific functional forms for the parameters of the generalized noise model can be derived. Additionally, a model is presented to described kinetic hole burning effects observed at low temperatures. Scaling laws are derived for these models that provide a connection with the generalized noise analysis.

Cardiac remodelling and myocardial contractility in patients with congestive heart failure treated with furosemide and enalapril.

Eleven patients with congestive heart failure class II-IV (NYHA) caused by ischemic heart disease were studied before and three months after adding enalapril to their treatment with furosemide. After an infarction the heart dilates gradually, mainly as a result of slippage of myocardial fiber bundles. It is known that the addition of an ACE-inhibitor to the medical treatment unloads the heart and gradually, within a period of 3 months, reduces heart size. Objectives of this study were to demonstrate remodelling by recording diastolic pressure-volume relations before and after treatment. The study addresses the question of whether regression of dilation, induced by the ACE-inhibitor treatment, improves the oxygen supply-demand ratio and, as a result, the contractility of the heart muscle. Treatment resulted in a reduction of vascular resistance (1479 to 1182 dyn.s.cm-5, p less than 0.05) and of the left ventricular end-diastolic (130 to 108 ml per m2 body surface area, p less than 0.05) and end-systolic (102 to 81 ml per m2 body surface area, p less than 0.01) volume index. The slope of the end-systolic pressure-volume relation, measured using vena cava occlusion and beat-to-beat recording of pressure and volume loops, remained unchanged. Indices of oxygen-supply demand ratio such as a drop of ejection fraction during exercise and parameters of active diastolic relaxation also did not change. Addition of an ACE-inhibitor induces regression of ventricular dilation, but no indications were found that it improves the condition of the cardiac muscle.