Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

Carbohydrates protect protein against abiotic fragmentation by soil minerals.

The degradation and turnover of soil organic matter is an important part of global carbon cycling and of particular importance with respect to attempts to predict the response of ecosystems to global climate change. Thus, it is important to mechanistically understand the processes by which organic matter can be degraded in the soil environment, including contact with reactive or catalytic mineral surfaces. We have characterized the outcome of the interaction of two minerals, birnessite and kaolinite, with two disaccharides, cellobiose and trehalose. These results show that birnessite reacts with and degrades the carbohydrates, while kaolinite does not. The reaction of disaccharides with birnessite produces Mn(II), indicating that degradation of the disaccharides is the result of their oxidation by birnessite. Furthermore, we find that both sugars can inhibit the degradation of a model protein by birnessite, demonstrating that the presence of one organic constituent can impact abiotic degradation of another. Therefore, both the reactivity of the mineral matrix and the presence of certain organic constituents influence the outcomes of abiotic degradation. These results suggest the possibility that microorganisms may be able to control the functionality of exoenzymes through the concomitant excretion of protective organic substances, such as those found in biofilms.

Assessing Coupled Protein Folding and Binding Through Temperature-Dependent Isothermal Titration Calorimetry.

Broad interest in the thermodynamic driving forces of coupled macromolecular folding and binding is motivated by the prevalence of disorder-to-order transitions observed when intrinsically disordered proteins (IDPs) bind to their partners. Isothermal titration calorimetry (ITC) is one of the few methods available for completely evaluating the thermodynamic parameters describing a protein-ligand binding event. Significantly, when the effective ΔH° for the coupled folding and binding process is determined by ITC in a temperature series, the constant-pressure heat capacity change (ΔCp) associated with these coupled equilibria is experimentally accessible, offering a unique opportunity to investigate the driving forces behind them. Notably, each of these molecular-scale events is often accompanied by strongly temperature-dependent enthalpy changes, even over the narrow temperature range experimentally accessible for biomolecules, making single temperature determinations of ΔH° less informative than typically assumed. Here, we will document the procedures we have adopted in our laboratory for designing, executing, and globally analyzing temperature-dependent ITC studies of coupled folding and binding in IDP interactions. As a biologically significant example, our recent evaluation of temperature-dependent interactions between the disordered tail of FCP1 and the winged-helix domain from Rap74 will be presented. Emphasis will be placed on the use of publically available analysis programs written in MATLAB that facilitate quantification of the thermodynamic forces governing IDP interactions. Although motivated from the perspective of IDPs, the experimental design principles and data fitting procedures presented here are general to the study of most noncooperative ligand binding equilibria.

Role of Ordered Proteins in the Folding-Upon-Binding of Intrinsically Disordered Proteins.

In this work, we quantitatively investigate the thermodynamic analogy between the folding of monomeric proteins and the interactions of intrinsically disordered proteins (IDPs). Motivated by the hypothesis that similar hydrophobic forces guide both globular protein folding and also IDP interactions, we present a unified experimental and computational investigation of the coupling between the folding and binding of the intrinsically disordered tail of FCP1 when interacting with the cooperatively folding winged-helix domain of Rap74. Our calorimetric measurements quantitatively demonstrate the significance of hydrophobic interactions for this binding event. Our computational studies indicate that IDPs relieve frustration at the surface of ordered proteins to generate a minimally frustrated complex that is strikingly similar to a globular monomeric protein. In summary, these results not only quantify the thermodynamic forces driving disordered protein interactions but also highlight the role of ordered proteins for IDP function.

Carbon-Detected (15)N NMR Spin Relaxation of an Intrinsically Disordered Protein: FCP1 Dynamics Unbound and in Complex with RAP74.

Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and as such exist as highly dynamic ensembles in solution. We present two (13)C-direct detection experiments for the measurement of (15)N NMR spin relaxation called the CON(T1)-IPAP and CON(T2)-IPAP that quantify backbone dynamics on a per-residue basis for IDPs in solution. These experiments have been applied to the intrinsically disordered C-terminal of FCP1, both free in solution and while bound to the RAP74 winged-helix domain. The results provide evidence that most of FCP1 remains highly dynamic in both states, while the 20 residues forming direct contact with RAP74 become more ordered in the complex. Parallel analysis of RAP74 backbone (15)N NMR spin relaxation reveals only very limited ordering of RAP74 upon FCP1 binding. Taken together, these data show that folding-upon-binding is highly local in this system, with disorder prevailing even in the complex.

The disordered C-terminus of the RNA polymerase II phosphatase FCP1 is partially helical in the unbound state.

Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and yet retain critical functions. Recycling of RNA Polymerase II after transcription is promoted by an interaction between the winged helix domain of RAP74, a component of the general transcription factor IIF (TFIIF), and the C-terminus of the TFIIF-associating CTD phosphatase (FCP1). Sixteen residues from the C-terminus of FCP1 form an α-helix in the complex, but the protein is otherwise agreed in the literature to be intrinsically disordered. Here we show through CD and recently developed carbon-detected NMR that, although FCP1 is intrinsically disordered, the above 16 residues composing the RAP74 binding surface form nascent α-helical structure in the unbound state. We further show retention of general FCP1 disorder and the nascent helical content in HeLa extract, establishing cellular relevance. The conformational bias observed leads to a mechanistic proposal for FCP1's transition from a disordered ensemble to an ordered conformation upon binding.