Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the single-molecule level have been developed. Advances in single-molecule fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.

Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.