Biology subjects

Luan, Q.

Publications and source records attributed to Luan, Q..

3 recordsLinked to original sources

bioRxiv2026

Autotransporter folding avoids a kinetic trap during vectorial translocation across the bacterial outer membrane

Autotransporter proteins are major virulence factors in Gram-negative pathogens, yet how they fold during secretion remains incompletely understood. A longstanding puzzle is why pertactin folds and is secreted in vivo within minutes but refolds in vitro over hours to days. We introduce BEAM, a multiscale framework that learns slow collective variables from coarse-grained simulations to guide all-atom enhanced sampling. Applied to a C-terminal segment of the pertactin passenger domain from Bordetella pertussis, BEAM achieved four- to six-fold greater conformational coverage than traditional collective-variable-guided adaptive sampling or unbiased molecular dynamics. The resulting free-energy landscape revealed a compact, non-native intermediate accessible in bulk solution but geometrically incompatible with vectorial translocation across the outer membrane. Kinetic simulations show that access to this intermediate slows folding, whereas excluding it produces rapid, in vivo-like kinetics. Together, these results explain how vectorial secretion accelerates pertactin folding by excluding an off-pathway kinetic trap. More broadly, BEAM provides a multiscale strategy for revealing hidden conformational states at atomic resolution.

biophysics↗

bioRxiv2026

Microfluidic Platform for Drug Response Profiling in NSCLC Patient-Derived Organoids

Tumor models that recapitulate 3D architecture are essential for understanding how cellular organization and microenvironmental interactions govern therapeutic response in human cancers. Here, we developed a microfluidic microphysiological system that enables controlled and scalable culture and drug testing of non-small cell lung cancer spheroids and patient-derived organoids. The platform integrated U-shaped microwells with dual-channel loading to support de novo spheroid formation, efficient trapping of pre-formed spheroids, and loading of intact organoids with reduced size heterogeneity. Tumor spheroids and organoids maintained high viability and structural integrity during long-term on-chip culture, and constrained microscale confinement produced ellipsoidal geometries that deviate from idealized spherical assumptions. Baseline genotype-dependent responses to KRAS G12C and EGFR inhibitors were preserved across agarose and microfluidic formats, establishing a validated reference state. Building on this baseline, fibroblast- and endothelial-derived cues consistently attenuated responses to targeted therapies across conditioned media, mixed co-culture, and spatially organized configurations. Resistance phenotypes converged on a dominant role for paracrine signaling, while increasing architectural complexity primarily enhanced morphological fidelity rather than altering therapeutic response. These findings establish a microphysiological framework that decouples tumor-intrinsic drug sensitivity from microenvironment-mediated modulation, enabling the systematic evaluation of paracrine resistance mechanisms in NSCLC.

bioengineering↗

bioRxiv2024

Discovery of an on-pathway protein folding intermediate illuminates the kinetic competition between folding and misfolding

Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. As protein length increases, the competition between off-pathway misfolding and on-pathway folding likewise increases, creating a more complex energy landscape. Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa {beta}-helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). The existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump "denaturant challenge" assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N-terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a two-step, C-to-N-terminal model for P.69T folding: folding initiates in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N-terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.

biophysics↗