Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions

Estrogens, selective estrogen receptor modulators, and a selective estrogen receptor down-regulator inhibit endothelial production of tissue factor pathway inhibitor 1

<p>Abstract</p> <p>Background</p> <p>Hormone therapy, oral contraceptives, and tamoxifen increase the risk of thrombotic disease. These compounds also reduce plasma content of tissue factor pathway inhibitor-1 (TFPI), which is the physiological inhibitor of the tissue factor pathway of coagulation. The current aim was to study if estrogens and estrogen receptor (ER) modulators may inhibit TFPI production in cultured endothelial cells and, if so, identify possible mechanisms involved.</p> <p>Methods</p> <p>Human endothelial cell cultures were treated with 17β-estradiol (E2), 17α-ethinylestradiol (EE2), tamoxifen, raloxifene, or fulvestrant. Protein levels of TFPI in cell media and cell lysates were measured by an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by quantitative PCR. Expression of ERα was analysed by immunostaining.</p> <p>Results</p> <p>All compounds (each in a concentration of 10 nM) reduced TFPI in cell medium, by 34% (E2), 21% (EE2), 16% (tamoxifen), and 28% (raloxifene), respectively, with identical inhibitory effects on cellular TFPI levels. Expression of TFPI mRNA was principally unchanged. Treatment with fulvestrant, which was also associated with down-regulation of secreted TFPI (9% with 10 nM and 26% with 1000 nM), abolished the TFPI-inhibiting effect of raloxifene, but not of the other compounds. Notably, the combination of 1000 nM fulvestrant and 10 nM raloxifene increased TFPI secretion, and, conversely, 10 nM of either tamoxifen or raloxifene seemed to partly (tamoxifen) or fully (raloxifene) counteract the inhibitory effect of 1000 nM fulvestrant. The cells did not express the regular nuclear 66 kDa ERα, but instead a 45 kDa ERα, which was not regulated by estrogens or ER modulators.</p> <p>Conclusion</p> <p>E2, EE2, tamoxifen, raloxifene, and fulvestrant inhibited endothelial production of TFPI by a mechanism apparently independent of TFPI transcription.</p