In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the Nδ1 atom of histidine and both the backbone N–H and side-chain Oγ-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of 2hJNN HBCs supports that Thr N–H···Nδ1 His H-bonds within internal repeats are stronger (∼4 Hz) than in the solvent exposed C-terminal AR (∼2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant 2hJ and 1hJ couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in 2hJNN, attributed to optimization of the Val N–H···Nδ1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.
Designed helical repeats (DHRs) are modular helix-loop-helix-loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.
A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N‐ vs. C‐terminal) interfacial coupling free‐energies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available at https://github.com/barricklab-at-jhu/Ising_programs.
A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N-versus C-terminal) interfacial coupling freeenergies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available at https://github.com/barricklab-at-jhu/Ising_programs. 3
Hydrogen bonds (H-bonds) are generally weak (< 20 kJ/mol) but ubiquitous interactions that determine in large part the cooperative folding and selfassembly of biological macromolecules in aqueous solution. Although important, most H-bonds are inferred indirectly and modeled using theoretical idealized geometry, therefore leaving instances of bifurcated or strained H-bonds undetected. Here, we present direct solution NMR evidence for an unusual bifurcated H-bonding arrangement comprising threonine Og-H and N-H donor protons and histidine acceptor Nd1 within the conserved TXXH alpha-helix N-capping motif of consensus ankyrin repeat (AR) proteins. The threonine N-H/Nd1 histidine H-bonds were detected in the TXXH motif of three and four-repeat AR proteins via 2h J NN hydrogen bond scalar coupling (HBC) NMR correlation experiments. Comparison of the 2h J NN couplings and histidine pK a values demonstrated that the N-H/Nd1 H-bonds of internal AR ( 2h J NN~4 Hz, pK a < 3) were stronger than those of the solvent-exposed C-terminal AR ( 2h J NN~2 Hz, pK a~5 .7). In addition to the N-H/Nd1 2h J NN couplings, we also observed distinct 1h J HN HBCs connecting the threonine Og-H and histidine Nd1 within the TXXH motif of internal ARs. Together, the 2h J NN and 1h J HN HBCs supported that a single histidine Nd1 acceptor had two H-bond donors. Accordingly, two-bond reciprocal isotope shifts were detected between the threonine N-H and Og-H protons in solvents with mixed H 2 O/D 2 O composition. Finally, a threonine to valine replacement, which eliminated the Og-H/Nd1 H-bond and resulted in a destabilized protein, lead to a relaxed valine N-H/Nd1 histidine interaction with enhanced 2h J NN (~5.2 Hz) likely owing to an optimized bonding geometry. Overall, our results provide new insight into the H-bonding ability of histidine and a challenging benchmark for the calculation of HBCs and their relation to H-bond energetics. Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. We show that methionine oxidation dramatically increases the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction that has emerged in recent years. We have performed a structural bioinformatics analysis of the PDB and CSD and found that interactions between DMSO (a small molecule analog of methionine sulfoxide (MetOx)) and aromatic groups are prevalent, even compared to methioninearomatic interactions. We then performed quantum mechanical calculations and found that DMSO-aromatic interactions are substantially strengthened compared to DMS-aromatic, especially in the cases of tyrosine and tryptophan which are able to donate a hydrogen bond to the sulfonyl oxygen. The relative bond energies correlate roughly with the relative abundance of phenylalaninetyrosine-and tryptophan-DMSO interactions found in the PDB. Through protein-stability experiments we show that oxidation of methionine w...
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