Increase in conformational entropy upon barnase-barstar high affin-

The NMR-relaxation measurements [17] of the backbone amide reveal that on average the changes in motion of the polypeptide backbone of barnase and barstar upon for- mation of the complex is small and reveals a general restriction of motion of the amide N-H bonds upon complex formation. The response of the individual sites is however somewhat heterogeneous (Figure5.12).

While the changes in motion of the backbone is small, the motions of the methyl-bearing amino acid side chains of both barnase and barstar increase upon complex formation (<O2_axis >=−0.096±0.012; n=90).

Figure 5.8: The 15_{N backbone amide chemical shifts obtained for free barnase (top}

panel) and free barstar (bottom panel) using triple resonance experiments match with previously published assignments.

Figure 5.9: The13_{C-methyl chemical shifts obtained for free barnase (left panel) and}

free barstar (right panel) match with previously published assignments.

Comparison of 13_{C-relaxation measured O}2

axis with previous 2H-relaxation

based measurements for free barnase and complexed barnase. The agree- ment between the methyl dynamics measurement for complexed barnase in this study to that measured previously is poor. This disagreement is a result of inac- curate characterization of macromolecular tumbling in the previous work where the molecular tumbling time was estimated based on the shape and hydrodynamic ra- dius of the complex. It is however important to note here that the correlation of mea- sured methyl dynamics in free barnase agrees reasonably well with the previous work because the molecular tumbling time for the free barnase was characterized using standard backbone NMR relaxation experiments. However, the measured methyl dynamics in complexed barnase differs significantly due to the inaccurate macromolecular tumbling estimate (Figure5.13). Hence, the observed increase in side chain dynamics differs from that described in the previous study of free and complexed barnase.

Figure 5.10: The15_{N backbone amide chemical shifts assignment (top panel) of bar-}

nase and barstar were assigned in the complexed state using triple resonance ex- periments. (bottom panel) The13_{C-methyl chemical shifts assignments obtained for}

barnase in the complex matched with previously published results. The 13_C-methyl

chemical shift of barstar in the complex were assigned. Residues which were over- lapped were not used for any of the analysis.

Figure 5.11: The 13_{C-aromatic chemical shifts of CD1 of Phe&Tyr and CE3 of Trp}

Figure 5.12: The changes in motion of the15_N−1_{Hamide bond are mapped onto the}

structure of the barnase-barstar complex (left panel) and indicate that the changes are on average small. The changes in motion of the individual sites in both barnase and barstar are indicated as a bar graph on the right. Some of the backbone amides in both barnase and barstar do not show any change in order parameter, these are not

indicated on the bar graph plot. PDB code barnase-barstar complex: 1BRS

of methyl-bearing side chains to changes in conformational entropy [14,107] indicates that the conformational entropy (−T∆Scon f) of barnase and barstar is increased by 18.5 kcal mol−1 upon complex formation (i.e. −T∆Scon f = -18.5 kcal mol−1) and therefore contributes favourably to the high affinity binding free energy. The distribution of the dynamical response of side chains to binding is also complex. Methyl-bearing residues of barnase at the interface experience small changes upon binding (< O2_axis|inter f ace >=

Figure 5.13: (A) The correlation of methyl order parameters for free barnase from the previous2_{H-relaxation measurement and the}13_{C-relaxation measurement in this study}

agree reasonably well (R2 _{= 0.8). (B) Poor correlation (R}2 _{= 0.4) of the methyl order}

parameters of barnase in the complexed state is observed between the2_{H- and}13_C-

relaxation studies due to the inaccurate macromolecular tumbling characterization in the previous2_{H-relaxation study.}

0.006±0.003; n=5) while those removed from the interface show a comparatively larger increase in motional amplitude (< O2_axis|remote >= −0.074±0.010; n=45). Similarly, the methyl-bearing residues of barstar at the interface also experience small changes upon binding (< O2_axis|inter f ace >= 0.01±0.005; n=3) while those removed from the interface show an even larger increase in motional amplitude than their counterparts in barnase (<O2_axis|remote>=−0.123±0.012; n=37) (Figure5.14).

The barnase-barstar interface is particularly enriched in aromatic amino acid residues. Using the recently introduced isotopic labeling strategy that eliminates a variety of technical issues (Chapter 3) [26], we were able to characterize the fast motion of 5

Figure 5.14: (A) Cartoon depiction of the barnase-barstar complex. (B) The changes in motion of the methyl-bearing amino acid side chains (∆O2_axis) in barnase and barstar upon complex formation iare mapped on to the structure indicating increase flexibility away from the interface and on average reduced flexibility at the interface of binding. The hydrophobic core of barnase which plays an important role in stabilitizing the beta-sheet surface involved in barstar and substrate binding displays reduced motion. Barstar displays an almost complete activation of dynamics indicating its high degree of optimization to bind barnase selectively. (C)&(D) represent the bargraph plot of

the changes in order parameter for barnase and barstar, respectively.

Phe, 6 Tyr and 4 Trp aromatic rings. The sensitivity of the fast aromatic ring mo- tions to binding of barstar to barnase mirrors that of the methyl-bearing side chains. We find that the motions of the aromatic side-chains at the interface, which are pre- dominantly involved in base-stacking and hydrogen bond interactions, are quenched (< O2_aro|inter f ace>=0.042±0.007; n=9), while the motions of those remote from the inter- face are activated (<O2_aro|remote>=−0.05±0.007; n=6) (Figure5.15).

Figure 5.15: (A) Cartoon depiction of the barnase-barstar complex. (B) The motions of the aromatic amino acid side chains (O2aro) are lowered in the interface of binding

while those remote from the binding interface show increased flexibility mirroring the behaviour of the methyl motions. PDB code barnase-barstar complex: 1BRS