The consequences of cavity creation on the folding landscape of a repeat protein depend upon context.

The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1-4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

Pediatric Supracondylar Humerus Fractures Can Be Safely Treated by Orthopaedic Surgeons With and Without Pediatric Fellowship Training.

BACKGROUND: The purpose of this study was to compare the outcomes of pediatric patients who were surgically treated for a supracondylar humerus fracture by pediatric fellowship-trained orthopaedic surgeons (PFT) to the outcomes of those surgically treated by orthopaedic surgeons without pediatric fellowship training (NPFT). We hypothesized that there would be no differences in patient outcomes. METHODS: A retrospective review of pediatric patients who underwent surgical treatment for a supracondylar humerus fracture with closed reduction and percutaneous pinning (CRPP) or open reduction and percutaneous pinning (ORPP) at a regional level 1 trauma center over a 5-year period was performed. Exclusion criteria were inadequate follow up or absence of postoperative radiographs. RESULTS: A total of 201 patients met the inclusion criteria. Pediatric-fellowship trained orthopaedic surgeons treated 15.9% of patients. There was no statistically significant difference in carrying angle, Baumann's angle, or lateral rotation percentage at final follow up between PFT and NPFT groups. There was no permanent neurovascular compromise in either group. Patients treated by NPFT were more likely to return to the operating room for pin removal. CONCLUSION: In this study, there was no difference in radiographic outcomes for patients with supracondylar humerus fractures surgically treated by either group. This suggests that pediatric supracondylar humerus fractures may be appropriately treated in communities without a pediatric-fellowship trained orthopaedic surgeon without compromised outcomes.Level of Evidence: III.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action.

Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries.

Solution-phase and solid-phase parallel synthesis and high throughput screening have enabled biologically active and selective compounds to be identified at an unprecedented rate. The challenge has been to convert these hits into viable development candidates. To accelerate the conversion of these hits into lead development candidates, early assessment of the physicochemical and pharmacological properties of these compounds is being made. In particular, in vitro absorption, distribution, metabolism, and elimination (ADME) assays are being conducted at earlier and earlier stages of discovery with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. In this report, we present an eight-channel parallel liquid chromatography/mass spectrometry (LC/MS) system in combination with custom Visual Basic and Applescript automated data processing applications for high throughput early ADME. The parallel LC/MS system was configured with one set of gradient LC pumps and an eight-channel multiple probe autosampler. The flow was split equivalently into eight streams before the multiple probe autosampler and recombined after the eight columns and just prior to the mass spectrometer ion source. The system was tested for column-to-column variation and for reproducibility over a 17 h period (approximately 500 injections per column). The variations in retention time and peak area were determined to be less than 2 and 10%, respectively, in both tests. The parallel LC/MS system described permits time-course microsomal incubations (t(o), t5, t15, t30) to be measured in triplicate and enables estimations of t 1/2 microsomal stability. The parallel LC/MS system is capable of analyzing up to 240 samples per hour and permits the complete profiling up to two microtiter plates of compounds per day (i.e., 176 test substrate compounds + sixteen controls).

Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries.

Early determinations of pharmaceutical properties can serve as predictors of a compound's likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays. The SAGIAN core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA robotic arm. The HLM stability assay utilizes a Multimek 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes. This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.

Observation of significant signal voids in images of large biological samples at 11.1 T.

Proton MRI of large biological samples were obtained on an 11.1 T / 40 cm instrument. Images were obtained of a fixed human brain and a large piece of fresh beef. The proton MR images demonstrate severe distortions within these conductive samples, indicative of shortened electrical wavelengths and wave behavior within the sample. These observations have significant implications with respect to the continuing evolution of MR to higher magnetic field strengths on large samples, particularly on humans.