Dynamics of a De Novo Designed Three-Helix Bundle Protein Studied by15N,13C, and2H NMR Relaxation Methods

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Abstract
Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. α3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of α3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, α3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of α3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13Cα relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the α-helical regions of α3D, with increasing mobility at the ends of the α-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of α3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and β-branched amino acids. The time scale of motions associated with methyl-bearing side chains of α3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of α3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.