Engineering ClpS for selective and enhanced N-terminal amino acid binding.

One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface.

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].

Probing the mechanism of cyanobacterial aldehyde decarbonylase using a cyclopropyl aldehyde.

Cyanobacterial aldehyde decarbonylase (cAD) is a non-heme diiron oxygenase that catalyzes the conversion of fatty aldehydes to alkanes and formate. The mechanism of this chemically unusual reaction is poorly understood. We have investigated the mechanism of C1-C2 bond cleavage by cAD using a fatty aldehyde that incorporates a cyclopropyl group, which can act as a radical clock. When reacted with cAD, the cyclopropyl aldehyde produces 1-octadecene as the rearranged product, providing evidence for a radical mechanism for C-C bond scission. In an alternate pathway, the cyclopropyl aldehyde acts as a mechanism-based irreversible inhibitor of cAD through covalent binding of the alkyl chain to the enzyme.

Mechanistic insights from reaction of α-oxiranyl-aldehydes with cyanobacterial aldehyde deformylating oxygenase.

The biosynthesis of long-chain aliphatic hydrocarbons, which are derived from fatty acids, is widespread in Nature. The last step in this pathway involves the decarbonylation of fatty aldehydes to the corresponding alkanes or alkenes. In cyanobacteria, this is catalyzed by an aldehyde deformylating oxygenase. We have investigated the mechanism of this enzyme using substrates bearing an oxirane ring adjacent to the aldehyde carbon. The enzyme catalyzed the deformylation of these substrates to produce the corresponding oxiranes. Performing the reaction in D2O allowed the facial selectivity of proton addition to be examined by (1)H NMR spectroscopy. The proton is delivered with equal probability to either face of the oxirane ring, indicating the formation of an oxiranyl radical intermediate that is free to rotate during the reaction. Unexpectedly, the enzyme also catalyzes a side reaction in which oxiranyl-aldehydes undergo tandem deformylation to furnish alkanes two carbons shorter. We present evidence that this involves the rearrangement of the intermediate oxiranyl radical formed in the first step, resulting in aldehyde that is further deformylated in a second step. These observations provide support for a radical mechanism for deformylation and, furthermore, allow the lifetime of the radical intermediate to be estimated based on prior measurements of rate constants for the rearrangement of oxiranyl radicals.

Diffusion heterogeneity tensor MRI (?-Dti): mathematics and initial applications in spinal cord regeneration after trauma - biomed 2009.

Diffusion weighted magnetic resonance imaging (DWI) is a powerful tool for evaluation of microstructural anomalies in numerous central nervous system pathologies. Diffusion tensor imaging (DTI) allows for the magnitude and direction of water self diffusion to be estimated by sampling the apparent diffusion coefficient (ADC) in various directions. Clinical DWI and DTI performed at a single level of diffusion weighting, however, does not allow for multiple diffusion compartments to be elicited. Furthermore, assumptions made regarding the precise number of diffusion compartments intrinsic to the tissue of interest have resulted in a lack of consensus between investigations. To overcome these challenges, a stretched-exponential model of diffusion was applied to examine the diffusion coefficient and "heterogeneity index" within highly compartmentalized brain tumors. The purpose of the current study is to expand on the stretched-exponential model of diffusion to include directionality of both diffusion heterogeneity and apparent diffusion coefficient. This study develops the mathematics of this new technique along with an initial application in quantifying spinal cord regeneration following acute injection of epidermal neural crest stem cell (EPI-NCSC) grafts.