Transient On- and Off-Pathway Protein Folding Intermediate States Characterized with NMR Relaxation Dispersion.

The earliest events in the folding of a protein are in general poorly understood. We used NMR relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.

Modular Protein Ligation: A New Paradigm as a Reagent Platform for Pre-Clinical Drug Discovery.

Significant resource is spent by drug discovery project teams to generate numerous, yet unique target constructs for the multiple platforms used to drive drug discovery programs including: functional assays, biophysical studies, structural biology, and biochemical high throughput screening campaigns. To improve this process, we developed Modular Protein Ligation (MPL), a combinatorial reagent platform utilizing Expressed Protein Ligation to site-specifically label proteins at the C-terminus with a variety of cysteine-lysine dipeptide conjugates. Historically, such proteins have been chemically labeled non-specifically through surface amino acids.

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2) relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.

The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS.

Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N(2) ⇆ I(2) ⇆ 2U) process involving a dimeric intermediate (I(2)). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking.

From two-state to three-state: the effect of the P61A mutation on the dynamics and stability of the factor for inversion stimulation results in an altered equilibrium denaturation mechanism.

Factor for inversion stimulation (FIS) is a 22 kDa homodimeric protein found in enteric bacteria that is involved in the stimulation of certain DNA recombination events and transcription regulation of many genes. FIS has a central helix with a 20 degrees kink, which is only reduced by 4 degrees after a proline 61 to alanine mutation (P61A). This mutation appears to have little effect on FIS function, yet it is striking that proline 61 is highly conserved among fis genes.