Table 1. Evaluating the Consistency of the MD Simulations with Experimental Observations.

no.	experiment	experimental observation	observation from optimized force field (unoptimized force field)	comments
1a	CD/NMR of Im76–45 by itself	11%/21% helicity	15% helicity (unoptimized: 10%)	this is the only observation that was used to optimize the individual force fields
2a	CD of Spy:Im76–45, subtracting CD spectrum of Spy alone	40% helicity of Im76–45 while in complex with Spy	36% helicity of Im76–45 while in complex with Spy (unoptimized: 23%)	increased agreement after optimization
3a	CD of Spy:Im76–45	+29 pp change in Im76–45 helicity upon bindingb	+21 pp change in Im76–45 helicity upon binding (unoptimized: +13 pp)	calculated from observations 1 and 3; increased agreement after optimization
4a	NMR titrations of Spy:Im76–45	see Figure 2 for per-residue intensity decreases of Spy	similar patterns observed in the frequency of contact between Spy and Im76–45	see the Contact Maps section in Supporting Methods and Figure S8
5c	X-ray crystallography	51% of residues on the concave surface of Spy participate in Im76–45 binding, compared to 12% of residues in other regions	51% of residues on the concave surface of Spy participate in Im76–45 binding, compared to 2% of residues in other regions (unoptimized: 31%/2%)	See the Interaction Patterns section in Supporting Methods and Figure S7
6c	X-ray crystallography	Spy dimer twists by −9° upon Im76–45 binding, as defined by an interdomain dihedral angle	Spy dimer twists, on average, by −4.6° along this same dihedral angle while bound to Im76–45	intramolecular Spy force field is the same in the optimized and unoptimized simulations, Figure 4
7c	X-ray crystallography	flexible linker region (residues 47–57) more disordered upon Im76–45 binding	upon binding, per-residue fluctuations increased by ∼50% relative to the unbound state for several residues in the flexible linker region	see comment for observation 6, Figure 4
8d	kinetics of Im7 folding while bound to Spy	binding to Spy slows down both folding and unfolding of full-length Im76–45 by approximately an order of magnitude	binding to Spy slows down folding (by a factor of 2) and unfolding (by a factor of 5) of the Im76–45 substrate	network analysis only performed for the optimized simulations, Figure 5
9e	genetic selections to increase Spy chaperone activity	mutants either increase concave surface hydrophobicity, or potentially increase flexibility of linker region	binding occurs primarily within cradle, and flexible linker region increases motion in bound state	see Site-Specific Description of Chaperone–Substrate Interactions
10e	in vitro cross-linking of Spy and Im76–45	all cross-links indicate binding to concave surface	see item 5 above
11e	in vitro H/D exchange and proteolysis of Spy upon Im76–45 binding	several residues on Spy's concave surface are protected upon binding	Spy interacts with Im76–45 through its concave surface	see Site-Specific Description of Chaperone–Substrate Interactions

^aThis study.

^bpp = percentage points.

^cRef 11.

^dRef 13.

^eRef 9.