In recent years mass spectrometry-based covalent labeling techniques such as hydroxyl radical footprinting (HRF) have emerged as valuable structural biology techniques, yielding information on protein tertiary structure. These data, however, are not sufficient to predict protein structure unambiguously, as they provide information only on the relative solvent exposure of certain residues. Despite some recent advances, no software currently exists that can utilize covalent labeling mass spectrometry data to predict protein tertiary structure. We have developed the first such tool, which incorporates mass spectrometry derived protection factors from HRF labeling as a new centroid score term for the Rosetta scoring function to improve the prediction of protein tertiary structures. We tested our method on a set of four soluble benchmark proteins with known crystal structures and either published HRF experimental results or internally acquired data. Using the HRF labeling data, we rescored large decoy sets of structures predicted with Rosetta for each of the four benchmark proteins. As a result, the model quality improved for all benchmark proteins as compared to when scored with Rosetta alone. For two of the four proteins we were even able to identify atomic resolution models with the addition of HRF data.
Hydroxyl radical
protein footprinting (HRPF) is a powerful and
flexible technique for probing changes in protein topography. With
the development of the fast photochemical oxidation of proteins (FPOP),
it became possible for researchers to perform HRPF in their laboratory
on a very short time scale. While FPOP has grown significantly in
popularity since its inception, adoption remains limited due to technical
and safety issues involved in the operation of a hazardous Class IV
UV laser and irreproducibility often caused by improper laser operation
and/or differential radical scavenging by various sample components.
Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting
System. This platform delivers sample via flow injection
to a facile and safe-to-use high-pressure flash lamp with a flash
duration of 10 μs fwhm. Integrated optics collect the radiant
light and focus it into the lumen of a capillary flow cell. An inline
radical dosimeter measures the hydroxyl radical dose delivered and
allows for real-time compensation for differential radical scavenging.
A programmable fraction collector collects and quenches only the sample
that received the desired effective hydroxyl radical dose, diverting
the carrier liquid and improperly oxidized sample to waste. We demonstrate
the utility of the FOX Protein Footprinting System by determining
the epitope of TNFα recognized by adalimumab. We successfully
identify the surface of the protein that serves as the epitope for
adalimumab, identifying four of the five regions previously noted
by X-ray crystallography while seeing no changes in peptides not involved
in the epitope interface. The FOX Protein Footprinting System allows
for FPOP-like experiments with real-time dosimetry in a safe, compact,
and integrated benchtop platform.
Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo-dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.
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