Molecular Dynamics of the Anti-Fluorescein 4-4-20 Antigen-Binding Fragment. 2. Time-Resolved Fluorescence Spectroscopy

Abstract

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.

Molecular evolution of affinity and flexibility in the immune system

“…Increased protein rigidity upon binding has also been observed in earlier studies of 4-4-20 by Lim and colleagues (17,18) and in other Ab systems (2). Indeed, the positive correlation between protein rigidity and binding affinity is likely to be a general feature of molecular association processes (24).…”

“…We observe that the environment of the combining site becomes more flexible when the ligand is absent or when affinity decreases. The entropic changes that accompany binding disfavor complex formation, a phenomenon noted in several other studies (17,18,(20)(21)(22)35). We find that the induced-fit paradigm predominantly applies to less mature antibodies and is substituted by more of a lock-and-key description of binding as maturation proceeds.…”

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

“…Antibody͞hapten recognition in the 4-4-20͞fluorescein model system used in this study has been characterized extensively by structural (11,12), thermodynamic (13), kinetic (14), computational (15), spectroscopic (16), and mutagenic (17) means. The 4-4-20 affinity for the fluorescein-biotin (FL-bio) hapten (K d ϭ 0.7 Ϯ 0.3 nM in PBS) is near the affinity ceiling of the tertiary immune response.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…For CYP51A1 we have chosen 3LD6 structure and for POR 3ES9 structure from RCSB Protein Data Bank (Berman et al, 2000). The point mutations were prepared using "residue mutagenesis" procedure in Pymol [2]. Appropriate rotamers were chosen, taking into account neighboring amino acids, as not to significantly perturb the structure of the protein.…”

“…Requires exogenous fluorophore with high quantum yield CYP, GST Greinert et al, 1979Gut et al, 1985;Schwarz et al, 1993;Gorovits and Horowitz, 1995;Lim et al, 1995;Lakowicz, 1999 , 1999, 2001Kuznetsov et al, 2004;Shimada et al, 2005;Pearson et al, 2006;Archakov and Ivanov, 2011;Martin et al, 2015;Yablokov et al, 2017 Quartz crystal microbalance conductometric Davydov et al, 2013b;Spera et al, 2013 X-ray crystallography with traditional site-directed mutagenesis techniques to obtain answers to structural questions that might not be readily available by either technique alone. X-ray crystallography has also been useful for dissecting the protein-protein interactions of another CYP electron transfer partner, b 5 .…”