B doi:10.1016/S0022-2836(02)00801-X available online at http://www.idealibrary.com on w J. Mol. Biol. (2002) 322, 645–652 The Backbone Conformational Entropy of Protein Folding: Experimental Measures from Atomic Force Microscopy James B. Thompson1, Helen G. Hansma1, Paul K. Hansma1* and Kevin W. Plaxco2,3* 1Department of Physics The energy dissipated during the atomic force microscopy-based University of California, Santa mechanical unfolding and extension of proteins is typically an order of Barbara, CA 93106-9510, USA magnitude greater than their folding free energy. The vast majority of the “excess” energy dissipated is thought to arise due to backbone confor- 2Department of Chemistry and mational entropy losses as the solvated, random-coil unfolded state is Biochemistry, University of stretched into an extended, low-entropy conformation. We have investi- California, Santa Barbara, CA gated this hypothesis in light of recent measurements of the energy dis- 93106-9510, USA sipated during the mechanical unfolding of “polyproteins” comprised of 3Interdepartmental Program in multiple, homogeneous domains. Given the assumption that backbone Biomolecular Science and conformational entropy losses account for the vast majority of the energy Engineering, University of dissipated (an assumption supported by numerous lines of experimental California, Santa Barbara, CA evidence), we estimate that ,19(^2) J/(mol K residue) of entropy is lost 93106-9510, USA during the extension of three mechanically stable b-sheet polyproteins. If, as suggested by measured peak-to-peak extension distances, pulling proceeds to near completion, this estimate corresponds to the absolute backbone conformational entropy of the unfolded state. As such, it is exceedingly close to previous theoretical and semi-empirical estimates that place this value at ,20 J/(mol K residue). The estimated backbone conformational entropy lost during the extension of two helical poly- proteins, which, in contrast to the mechanically stable b-sheet polyproteins, rupture at very low applied forces, is three- to sixfold less. Either previous estimates of the backbone conformational entropy are significantly in error, or the reduced mechanical strength of the helical proteins leads to the rupture of a subsequent domain before full extension (and thus complete entropy loss) is achieved. q 2002 Elsevier Science Ltd. All rights reserved *Corresponding authors Keywords: configurational entropy; worm-like chain Introduction tain a wealth of information regarding the mechanical properties, folding kinetics and Atomic force microscopy (AFM) and optical thermodynamics of proteins, some of which has tweezers have been used to characterize the not been explored in detail. Here, for example, we mechanical properties and force-induced unfold- demonstrate that AFM-unfolding may provide an ing of a wide range of proteins.1–15 When applied experimental means of measuring the backbone to a protein comprised of multiple independent conformational entropy of an unfolded protein. folding units (domains), these pulling experiments The area under each peak in AFM-unfolding typically produce a saw-toothed force versus exten- force–extension curves represents the energy sion curve as the domains sequentially unfold and required to unfold a domain and to pull the extend. These pioneering pulling experiments con- unfolded state into an extended conformation (Figure 1). The energy dissipated during such a Abbreviations used: AFM, atomic force microscopy; mechanical unfolding event is, however, an order Ig, immunoglobulin domain. of magnitude greater than the typical free energy E-mail addresses of the corresponding authors: of folding of a single-domain protein. The dis- [email protected]; [email protected] crepancy is thought to arise due to the additional 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved 646 Backbone Conformational Entropy of Folding Figure 1. A diagram of the force- induced unfolding of a multi- domain protein. The area under the force–extension curve represents the energy dissipated during the pulling process. While a number of factors may contribute to this energy, the excellent fit of force– extension curves to the expected behavior of an inert, worm-like chain2,5,8 suggests that the loss of conformational entropy dominates. If this is true and the extension between subsequent rupture events is near completion (i.e. that the maximally extended state adopts one or a few conformations) the energy dissipated then provides an experimental means of determining the backbone conformational entropy change associated with protein folding. energy input required to extend the initially highly The five suitable polyproteins represent both disordered, random-coil unfolded state into one or predominantly sheet and predominantly helical a few near-fully extended conformations, a structures. Fernandez, Clarke and co-workers hypothesis supported by the observation that have used protein engineering to create homo- force–extension curves are well fit by models of geneous polyproteins consisting of multiple the entropic cost of extending an inert, worm-like repeats of identical, predominantly b-sheet chain.2,5,8 Here, we investigate this hypothesis in immunoglobulin (Ig) domains.6,7,9 Bustamante, the light of previous estimates of the backbone con- Dahlquist and co-workers, in turn, have used formational entropy of an unfolded polypeptide. solid-phase coupling to convert monomeric, helical T4 lysozyme into a covalent, disulfide-bonded polyprotein.10 Gaub and co-workers have engi- Results neered a multi-domain protein comprised of four homologous, structurally identical, predominantly Early protein pulling studies focused on natu- helical a-spectrin domains.4 Because each of the rally occurring multidomain proteins such as titin, well-characterized domains comprising them is tenascin and fibronectin.1–3,11 These proteins are structurally (and, with the exception of the comprised of multiple copies of two or three struc- a-spectrin polyprotein, mechanically and thermo- turally distinct domain types and thus their force– dynamically) identical with its neighbors, these extension curves reflect contributions from systems are ideally suited for detailed studies of structural units that vary in terms of both unfold- folding thermodynamics. ing free energy and extension length. This hetero- Fernandez, Clarke and co-workers have syn- geneity is evident in the observation that thesized and characterized the folding and sequential rupture peaks occur at increasing force mechanical unfolding of polyproteins comprised as the mechanically weakest domains break prior of either the 27th or 28th Ig domains (I27, I28) to mechanically stronger domains (e.g. see Figure from the human muscle protein titin.6,9 These 89 1A of Li et al.9). Because it is generally not possible residue, predominantly b-sheet domains rupture to correlate the individual features in a force– at applied forces of 204(^16) pN and extension curve with the unfolding and extension 257(^27) pN, respectively, reflecting their of a specific domain, it is difficult to account relatively high mechanical stability. The force– quantitatively for the energy dissipated in the extension curves observed upon their unfolding mechanical unfolding of these proteins. In order to during mechanical pulling are well fit by models circumvent this complication, several groups have of the entropy lost as a worm-like chain is employed protein engineering to produce “poly- extended.6,9 We have integrated the worm-like proteins” comprised of multiple copies of similar chain model and found that the unfolding and or identical domains.4,6,7,9,10,13,14 For these homo- extension these domains dissipates 390(^30) kJ/ geneous polyproteins, sequential rupture peaks mol and 460(^30) kJ/mol, respectively. If these occur at an approximately constant force, demon- energies arise predominantly due to chain entropy strating that their structural homogeneity trans- restrictions that are distributed evenly among the lates into mechanical homogeneity (e.g. see force-hidden residues in the domain (both con- Figures 1B and C, and 2 of Li et al.9). Five of these tour-length changes and inspection of the Ig struc- polyproteins are comprised of structurally well- ture suggest 75 residues are liberated upon characterized domains that rupture at measurably unfolding7), we find they correspond to confor- high forces. We have focused our investigations mational entropies of 17.4(^1.3) J/(mol K residue) on these well-defined systems. and 20.6(^1.3) J/(mol K residue), respectively. Backbone Conformational Entropy of Folding 647 Fernandez and co-workers have investigated the ordered extended state requires that the poly- issue of “force-hidden” residues by constructing peptide chain is pulled to nearly full extension. several mutant I27 polyproteins containing Consistent with this argument, the reported peak- unstructured penta-glycine loops.7 In two of the to-peak extension of the I27 domain, 24(^1) nm,6 three mutant proteins, the loops are inserted in is quite close to the ,26 nm we calculate for full inter-domain linker regions that are force-bearing extension. Similarly, the reported change in exten- and are thus extended prior to the unfolding of sion length upon the insertion of five glycine the first domain. Consistent with this suggestion, residues, ,1.9 nm,7 is consistent with the calcu- these insertions alter neither the contour length lated full extension of