A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins.

Orthogonal high-throughput thermal scanning method for rank ordering protein formulations.

A high-throughput thermal-scanning method to rank-order formulation conditions for therapeutic proteins is described. Apparent transition temperatures for unfolding and aggregation of four different proteins are determined using the dyes SYPRO Orange and thioflavin T (ThT) under a variety of buffer conditions. The results indicate that the ThT-based thermal scanning method offers several advantages over the previously described SYPRO Orange-based thermal scanning and allows rapid rank ordering of solution conditions relevant toward long-term storage of therapeutic molecules.

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC·PCNA·DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC·PCNA·DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC·PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polδ only one species converts to the RFC·PCNA·DNA·polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

Coupled motions in enzyme catalysis.

Recently, the hypothesis that protein motions are involved in enzymatic turnover has gained significant attention. We review cases where there is evidence that protein motions are rate-limiting in the overall catalytic cycle and examine experimental and theoretical evidence for how such motions enhance the probability of sampling the transition state configurations relative to the ground state. The impact of tunneling, the possible role of vibrational coupling and the value of conformational chemical landscapes are also scrutinized.

Surface sites for engineering allosteric control in proteins.

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR).

Efficient primer strand extension beyond oxadiazole carboxamide nucleobases.

Two oxadiazole carboxamide deoxyribonucleoside analogues are described that can be incorporated and efficiently extended by Taq DNA polymerase. The primer strand extension beyond oxadiazole nucleoside analogues occurs at rates similar to the values observed for the canonical Watson-Crick base pairs irrespective of the template nucleobase. These distinctive chemical effects in DNA polymerase extensions are attributed to the smaller size and unique electronic properties of the oxadiazole nucleobase.

Adaptive silver films for detection of antibody-antigen binding.

Antibody-antigen binding events at a monolayer protein concentration have been demonstrated on nanostructured adaptive silver films (ASFs) using surface-enhanced Raman scattering (SERS) and luminescence-based assays. It is shown that proteins stabilize and restructure the ASF to increase the SERS signal while preserving antigen-binding activity. Evidence for antibody-antigen binding on the ASF substrates is the distinct SERS spectral changes of the surface-bound antibody or antigen without special tags.

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs.

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases.