Kozlowski, N. | bioRxiv Science

bioRxiv2026

Combining CHARMM36m with OPC water improves accuracy

Atomistic simulations of intrinsically disordered proteins (IDPs) are notoriously sensitive to force field inaccuracies, either regarding the protein or the water model, yielding inaccurate observables such as compactness, secondary structure propensities, or kinetics. The currently most widely used IDP force fields are Amber99sb-disp (A99disp) and Charmm36m (C36m). A99disp includes a new water model and thus optimized both, the protein and the water interactions. In contrast, C36m used the Tip3p water model and optimized only protein interactions. In many cases, C36m+Tip3p underestimates radii of gyration compared to FRET or SAXS experiments. Such overly compact structural ensembles are believed to arise from an imbalance between protein-protein, protein-water, and water-water interactions, which might be due to Tip3p inaccuracies. Here, we aim at re-balancing these interactions by combining C36m with the Optimal Point Charge (OPC) water model. Recently, this C36m+OPC combination showed improved accuracy for the disordered domain of the measles virus nucleoprotein. Here we present a systematic assessment, comparing C36m+OPC, C36m+Tip3p, C36m+Tip4p, C22*, A03ws, A99sb-ws, and A99disp for five IDPs, as well as a subset of those for five globular proteins, a set of disordered AGQ-repeat peptides, and the fast folding miniprotein CLN025. We compared extensive MD simulations (> 8.5 ms) with SAXS, NMR, circular dichroism, photo-induced electron transfer (PET), T-jump infrared spectroscopy, and X-ray crystallography measurements. We found that combining C36m with OPC improved accuracy over C36m+Tip3p for IDP ensembles without compromising its accuracy for globular proteins. While also the kinetics of the AGQ-peptides were more accurate for C36m+OPC, those of CLN025 folding were less accurate. Overall, C36m+OPC showed similar accuracy as A99sb-ws and A99disp, the latter is currently considered among the most accurate protein force fields.

biophysics↗

bioRxiv2019

TIMP-1 attenuates the development of cutaneous inflammation-induced hypersensitivity

Unresolved inflammation is a significant predictor for developing chronic pain, and targeting the mechanisms underlying inflammation offers opportunities for therapeutic intervention. During inflammation, matrix metalloproteinase (MMP) activity contributes to tissue remodeling and inflammatory signaling through proteolytic maturation of cytokines. MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs) 1-4. TIMP-1 and -2 have known roles in pain, but only in the context of MMP inhibition. However, TIMP-1 also has receptor-mediated cell signaling functions that are not well understood. Here, we examined how TIMP-1-dependent cell signaling impacted inflammatory hypersensitivity and ongoing pain. We found that hindpaw injection of complete Freunds adjuvant (CFA) increased keratinocyte-derived TIMP-1 that peaked 3 days following inflammation, when mechanical hypersensitivity began to emerge in WT mice. These data suggest that TIMP-1 expression inhibits the development of inflammatory hypersensitivity. To examine this possibility, we injected TIMP-1 knockout (T1KO) mice with a diluted CFA mixture to examine how subtle cutaneous inflammation affected behavioral hypersensitivity. T1KO mice exhibited rapid onset thermal and mechanical hypersensitivity at the site of inflammation that was absent or attenuated in WT controls. We also found that T1KO mice exhibited hypersensitivity in adjacent tissues innervated by different sets of afferents, and skin contralateral to the site of inflammation. Replacement of recombinant murine (rm)TIMP-1 alleviated hypersensitivity when administered at the site and time of inflammation. To examine the MMP-dependent and -independent mechanisms of rmTIMP-1, T1KO mice were administered full-length rmTIMP-1, the N-terminal region (TIMP-1(N)) with MMP-inhibitory properties, or the C-terminal region (TIMP-1(C)) that retains receptor signaling function. Each of the peptides prevented inflammatory hypersensitivity, suggesting that rmTIMP-1 acts through mechanisms that also include receptor-mediated cell signaling. We also found that hypersensitivity was neither due to genotype-specific differences in MMP-9 activity or expression, nor to differences in cytokine expression. Finally, to evaluate the potential clinical utility of TIMP-1, we administered rmTIMP-1 to WT mice and found that rmTIMP-1 prevented clonidine-induced conditioned place preference (e.g., ongoing pain) and inflammatory mechanical hypersensitivity. Collectively, our data suggest a novel role for TIMP-1 in the attenuation of inflammatory pain that occurs through previously uncharacterized cell signaling mechanisms.

neuroscience↗