A central challenge in protein biophysics is to decipher the structural features that give rise to the energetics of protein folding pathways and determine structural details of intermediates. A unique advantage of single-molecule force spectroscopy over classical thermodynamic methods of monitoring protein folding and unfolding transitions is that force spectroscopy effectively reduces the ensemble of possible conformational states to those whose unfolding trajectories share a common reaction coordinate experimentally controlled by the vector of applied force. Although the dimensionality of the energy landscape for folding is simplified relative to traditional spectroscopic methods that monitor folding in solution, force spectroscopy shares the limitation with classical methods in that it cannot directly decipher the structural properties of partially folded intermediates in folding pathways from the energetics. What is needed is a means by which to deduce intermediate state structure using an energy-to-structure translator, one that can provide models for partially folded intermediates that are commensurate with experimentally determined energetics when given structural coordinates of the native state and residue specific thermodynamic contributions to folding. The article by Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) leverages the transfer model, with single-molecule force spectroscopy performed in the presence of two osmolytes (sorbitol and TMAO), to decipher the structure of an on-pathway intermediate in the folding of a cysteine-free variant of T4 lysozyme (T4∗). Naturally occurring osmolytes are small organic molecules that regulate cell volume and protect cells against water stress conditions (extremes of temperature, pressure, desiccation, extracellular osmolality, or intracellular denaturants) encountered by the organism. The transfer model uses experimentally determined free energies of interaction between various osmolyte solutions and model compounds that mimic solvent-exposed peptide backbone and side-chain groups comprising the protein structure to derive thermodynamic predictions of osmolyte-driven protein folding and stabilization (2Auton M. Bolen D.W. Application of the transfer model to understand how naturally occurring osmolytes affect protein stability.Methods Enzymol. 2007; 428: 397-418Crossref PubMed Scopus (89) Google Scholar). A unifying principle of the transfer model is that the thermodynamic action of stabilizing osmolytes on the unfolded state is dominated by unfavorable interactions with the peptide backbone (3Bolen D.W. Baskakov I.V. The osmophobic effect: natural selection of a thermodynamic force in protein folding.J. Mol. Biol. 2001; 310: 955-963Crossref PubMed Scopus (555) Google Scholar), which drives denatured state contraction (4Qu Y. Bolen C.L. Bolen D.W. Osmolyte-driven contraction of a random coil protein.Proc. Natl. Acad. Sci. USA. 1998; 95: 9268-9273Crossref PubMed Scopus (270) Google Scholar) leading to increased secondary structure content before the folding transition (5Holthauzen L.M.F. Rösgen J. Bolen D.W. Hydrogen bonding progressively strengthens upon transfer of the protein urea-denatured state to water and protecting osmolytes.Biochemistry. 2010; 49: 1310-1318Crossref PubMed Scopus (64) Google Scholar). These properties of protecting osmolytes favor the formation of intermediate structure in folding pathways. In their study, Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) demonstrate that although the mechanical unfolding of T4∗ by constant velocity force rupture is unaffected by osmolytes, osmolytes do affect the lifetimes of folding and unfolding under constant force. The kinetics of unfolding are decelerated while folding is accelerated, a result consistent with kinetic measurements of chemical denaturation in the presence of protecting osmolytes in solution (6Russo A.T. Rösgen J. Bolen D.W. Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization.J. Mol. Biol. 2003; 330: 851-866Crossref PubMed Scopus (65) Google Scholar). Furthermore, the force-clamp measurements observe transient flickering into a state of intermediate extension between the unfolded and native states at constant low force, but, once T4∗ folds to the native state, the folding reaction is essentially irreversible. Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) conclusively demonstrate that, even at the single-molecule level, the action of osmolytes is primarily on the unfolded to intermediate state folding transition such that both sorbitol and TMAO differentially enhance the intermediate population before the collapse to the native fold. Taking advantage of the transfer model’s ability to predict the experimental effects of osmolytes on the thermodynamic stability of two-state folding proteins (7Auton M. Rösgen J. Bolen D.W. et al.Osmolyte effects on protein stability and solubility: a balancing act between backbone and side-chains.Biophys. Chem. 2011; 159: 90-99Crossref PubMed Scopus (182) Google Scholar), Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) utilize transfer free energies together with a computational method of parsing the known native structure into all possible contiguously folded regions to enumerate a small ensemble of partially folded states. While the folded elements of these states are extracted from the crystal structure, the unfolded elements are determined from steered molecular dynamic simulations in which the solvent accessible surface area of the protein groups is calculated as a function of chain extension. These surface areas are weighted according to the transfer free energies specific to each osmolyte and each residue type (2Auton M. Bolen D.W. Application of the transfer model to understand how naturally occurring osmolytes affect protein stability.Methods Enzymol. 2007; 428: 397-418Crossref PubMed Scopus (89) Google Scholar) and summed for each computed state of the ensemble. Using sound scientific deduction, Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) recognize that because different osmolytes have different interaction free energies with backbone and side-chain protein groups, the energetic response of each intermediate to different osmolytes will be unique. It is this very inference that provides the rationale for performing force spectroscopy with two or more osmolytes. Different types of solvent-exposed surface area in intermediates will have different energetic potentials to fold as well as distinct sensitivities to various osmolytes. Yet the underlying principle of backbone dominance in the energetics of osmolyte-driven protein folding supports the idea of a single unfolding pathway with a unique intermediate. It is conceivable that different osmolytes might alter the folding pathway through different intermediates due to the osmolyte-dependent balance of backbone and side-chain energetic contributions within the protein chain topology. However, Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) demonstrate that only a subset of the computed intermediate state structures have energetics that (1) are common to both sorbitol and TMAO, (2) fall within the physical constraints of the experimental change in contour length, and (3) are consistent with prior experimental observations made independently by others (8Cellitti J. Llinas M. Marqusee S. et al.Exploring subdomain cooperativity in T4 lysozyme I: structural and energetic studies of a circular permutant and protein fragment.Protein Sci. 2007; 16: 842-851Crossref PubMed Scopus (30) Google Scholar). Recently, the src SH3 domain was shown to have multiple unfolding pathways depending on the direction of applied force and the presence of urea and/or mutations (9Guinn E.J. Jagannathan B. Marqusee S. Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein.Nat. Commun. 2015; 6: 6861Crossref PubMed Scopus (63) Google Scholar). It would be valuable to utilize the methods employed by Motlagh et al. (1Motlagh H.M. Toptygin D. Hilser V.J. et al.Single-molecule chemo-mechanical spectroscopy provides structural resolution of protein folding intermediates.Biophys. J. 2016; 110: 1280-1290Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) to determine whether different force trajectories alter intermediate structure and whether different osmolytes can alter the folding pathway within defined vectors of applied force. Resolving such highly dynamic conformational states of proteins has implicit benefit for uncovering the mechanistic principles that determine protein function. For example, the transfer model has also been successfully implemented into algorithms that define thermodynamic subdomains of protein structures consistent with folding intermediates (10Porter L.L. Rose G.D. A thermodynamic definition of protein domains.Proc. Natl. Acad. Sci. USA. 2012; 109: 9420-9425Crossref PubMed Scopus (35) Google Scholar) and that decipher structural regions responsible for mutation-induced local unfolding and altered function in the platelet binding domain of von Willebrand factor (11Zimmermann M.T. Tischer A. Auton M. et al.Structural origins of misfolding propensity in the platelet adhesive von Willebrand factor A1 domain.Biophys. J. 2015; 109: 398-406Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). While the transfer model has room to improve, particularly with respect to the multicomponent solution physical chemistry needed to accurately quantify the chemical potentials specific to group transfer free energies (12Auton M. Bolen D.W. Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale.Biochemistry. 2004; 43: 1329-1342Crossref PubMed Scopus (189) Google Scholar), this application of the transfer model in conjunction with osmolyte-specific force spectroscopy is a novel advance in its incorporation of ensemble-based statistical thermodynamics with single-molecule methods of probing folding pathways to structurally resolve on-pathway intermediates.