Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein conformational entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in conformational dynamics as a proxy for corresponding changes in conformational entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding conformational entropy is linearly related to the change in the overall binding entropy. This indicates that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association.

The physical basis for high affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently employed changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an “entropy meter” employing an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin are surprisingly non-complementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

As the primary intracellular calcium sensor, calmodulin (CaM) regulates numerous and diverse proteins. Several mechanisms, including tissue specific expression, localization and sequestration, work in concert to limit the total number of available targets of calmodulin within a cell. While the free energies of binding of calmodulin-binding domains of regulated proteins by CaM have been shown to be highly similar, they result from vastly different enthalpic and entropic contributions. Here, we report the backbone and side-chain methyl dynamics of calcium-activated calmodulin in complex with a peptide corresponding to the CaM binding domain of calmodulin kinase kinase, along with the thermodynamic underpinnings of complex formation. The results show a considerable reduction in side-chain mobility throughout CaM upon binding the CaMKKα peptide which is consistent with the enthalpically driven nature of the binding. Site specific comparison to another kinase-derived peptide complex with similar thermodynamic values, reveals significant differences in dynamics largely localized to the hydrophobic binding sites.Calmodulin is a highly conserved protein, capable of binding and regulating numerous proteins in response to increased levels of intracellular calcium (1,2). Calcium-saturated calmodulin (CaM) is the dominant regulatory species though there are several cases where apocalmodulin is found to bind target proteins. Current estimates indicate well over 300 proteins contain a CaM binding motif (3). Critical among these are kinases which subsequently regulate gene transcription, muscle contraction, transport, and other cellular processes (4). Calcium/ calmodulin-dependent protein kinase kinase (CaMKK) acts to enhance the activity of downstream CaM-dependent kinases (types I and IV) by phosphorylation of a single Thr in the so-called activation loop (5-7). CaMKI has recently been shown to function within the ERK signaling pathway (8) and CaMKI and CaMKIV differentially activate the transcription factors CREB, CREM, and ATF-1 (9-11). Two isoforms of CaMKK are known (α and β) each with abundant expression in neuronal, although CaMKKβ also has low level expression in the † Supported by NIH research grant DK 39806. *To whom correspondence should be addressed. Email: wand@mail.med.upenn.edu. Contact Information: Professor A. Joshua Wand, Department of Biochemistry & Biophysics University of Pennsylvania Philadelphia, PA 19104-6059, Tel: 215-573-7288, Fax: 215-573-7290, E-mail: wand@mail.med.upenn.edu 1 Abbreviations used: CaM, calcium-saturated calmodulin; CaMKK, calcium/calmodulin-dependent protein kinase kinase; CaMKKαp, peptide based on the CaMKKα calmodulin-binding domain with sequence CaMKKα sequence: GSVKLIPSWTTVILVKSMLRKRSFGNPF; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; NOE, { 1 H}-15 N nuclear Overhauser effect; and , Lipari-Szabo squared generalized order parameters for the methyl group symmetry axis and amide N-H bond, respectively; PCR, polymerase chain reaction; rmsd, root m...

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of nonspecific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAbdependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.

Adequate stability, manufacturability, and safety are crucial to bringing an antibody-based biotherapeutic to the market. Following the concept of holistic in silico developability, we introduce a physicochemical description of 91 market-stage antibody-based biotherapeutics based on orthogonal molecular properties of variable regions (Fvs) embedded in different simulation environments, mimicking conditions experienced by antibodies during manufacturing, formulation, and in vivo. In this work, the evaluation of molecular properties includes conformational flexibility of the Fvs using molecular dynamics (MD) simulations. The comparison between static homology models and simulations shows that MD significantly affects certain molecular descriptors like surface molecular patches. Moreover, the structural stability of a subset of Fv regions is linked to changes in their specific molecular interactions with ions in different experimental conditions. This is supported by the observation of differences in protein melting temperatures upon addition of NaCl. A DEvelopability Navigator In Silico (DENIS) is proposed to compare mAb candidates for their similarity with market-stage biotherapeutics in terms of physicochemical properties and conformational stability. Expanding on our previous developability guidelines (Ahmed et al. Proc. Natl. Acad. Sci. 2021, 118 (37), e2020577118), the hydrodynamic radius and the protein strand ratio are introduced as two additional descriptors that enable a more comprehensive in silico characterization of biotherapeutic drug candidates. Test cases show how this approach can facilitate identification and optimization of intrinsically developable lead candidates. DENIS represents an advanced computational tool to progress biotherapeutic drug candidates from discovery into early development by predicting drug properties in different aqueous environments.

No abstract

To cite this article: Ram P Vanam, Michael A Schneider & Michael S Marlow (2015) Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity, mAbs, 7:6, 1118-1127, DOI: 10.1080DOI: 10. /19420862.2015 An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.