Elimination of the C-cap in ubiquitin - structure, dynamics and thermodynamic consequences

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.     

Codon preference optimization increases heterologous PEDF expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ～3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ～4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ～3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.     

Pigment Epithelium-Derived Factor (PEDF) Peptide Eye Drops Reduce Inflammation,Cell Death and Vascular Leakage in Diabetic Retinopathy in Ins2Akita Mice

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60–77 (P60) and neuroprotective PEDF78–121 (P78) derivatives were applied to Ins2^Akita mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1–4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.     

Cooperativity of complex salt bridges

The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge is more than the sum of the energies of individual pairs. In one case, it was reported that complex salt bridge formation is anti-cooperative. To resolve these different findings, we performed analysis of the geometries of salt bridges in a representative set of structures from the PDB and found that over 87% of all complex salt bridges anchored by Arg/Lys have a geometry such that the angle formed by their Calpha atoms, Theta, is <90 degrees . This preferred geometry is observed in the two reported instances when the energetics of complex salt bridge formation is cooperative, while in the reported anti-cooperative complex salt bridge, Theta is close to 160 degrees . Based on these observations, we hypothesized that complex salt bridges are cooperative for Theta < 90 degrees and anti-cooperative for 90 degrees < Theta < 180 degrees . To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Theta = 150 degrees into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). Experimentally derived stabilities of the ADA2h variants allowed us to show that the complex salt bridge in ADA2h is anti-cooperative.     

Bochdalek Hernia As A Cause Of Intermittent AV Block

Congenital diaphragmatic hernia is very rare cause of AV block. We report such a patient with sick sinus node syndrome and previous AAIR pacemaker implantation, in which intermittent AV block was diagnosed by 24-hours ECG monitoring and upgrade of pacing system to DDDR was suggested.