Two states or not two states: Single-molecule folding studies of protein L

Abstract

Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ∼7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

Single-molecule FRET methods to study the dynamics of proteins at work

“…A different set of trajectory-analysis methods starts with a kinetic model, the parameters of which are inferred from the data using a statistical approach based on maximum-likelihood estimation using, for example, Hidden Markov Models (HMM) [60]. HMM has been demonstrated to be a powerful tool for analyzing smFRET trajectories [61] (Figure 1c). A limitation of HMM is the need to assume a priori the number of states involved in the dynamics.…”

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered

“…These spectroscopic methods can identify multiple, simultaneously populated conformational states. However, when more than two states are observed, information on the pathways connecting different states and, importantly, the order in which they are populated must come from single-molecule measurements (Aviram et al, 2018;Chung et al, 2012;Kim and Chung, 2020;Schuler et al, 2002).…”

Single‐Molecule FRET of Membrane Transport Proteins

“…For the last two decades, [1] smFRET techniques have been extensively used to study the properties of molecular machines, [2] intrinsically disordered proteins (IDPs),[ 3 , 4 , 5 , 6 , 7 , 8 ] protein folding processes,[ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ] protein‐ligand[ 17 , 18 , 19 ] and protein‐nucleic acid interactions,[ 20 , 21 , 22 , 23 ] as well as other structure‐function relationships and dynamic processes. [ 24 , 25 , 26 ] SmFRET is a particularly powerful and versatile tool to gain molecular and mechanistic insights because of its high spatial resolution (2–10 nm) combined with a wide range of accessible timescales (ns‐minutes).…”