Side-Chain Dynamics of an α-Helical Polypeptide Monitored by Fluorescence

Abstract

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.

Long Range Polymer Chain Dynamics Studied by Fluorescence Quenching

“…To date, the FBM has been applied to the analysis of the monomer and excimer decays acquired with several polymers randomly labeled with pyrene. 35,37,67,68,[72][73][74][75] Table 2. Chemical structure of the polystyrenes and poly(N-isopropylacrylamide) randomly labeled with pyrene.…”

Intramolecular Excimer Formation and Delayed Fluorescence in Sterically Constrained Pyrene Dimers

“…The intention is to balance the p-stacking tendency of the aromatic units, as evidenced by excimer fluorescence, against the inhibitory effects of the side chains. Such studies form an essential part of the protein-folding problem [18] and might lead to the design of nextgeneration foldamers [19] that possess predetermined electronic properties. With respect to these properties, it is well known [16] that pyrene displays delayed fluorescence by way of triplet-triplet annihilation.…”

Contraction and Collapsing Kinetics of Single Synthetic Polymer Chains at Small Quench Depths

“…For pyrene-labeled polymer chains, the latter is determined by the chain mobility and local pyrene concentrations. [86,87] Martinho and Duhamel et al [19,21,81,82] employed equilibrium excimer fluorescence to investigate the coil-toglobule transition thermodynamics of individual chains and a blob model was proposed accordingly. [86,87] The fluorescence technique enabled them to study the equilibrium collapsing transitions of short chains when labeled with appropriate fluorescent dyes.…”

“…(1) In protein folding kinetic studies, the exponential fitting of time-dependent fluorescence (intrinsic fluorescence of tryptophan residues, added ANS probes, FRET) changes has been typically employed, and the results generally agree with the time-dependence size changes. [92] (2) In a series of reports by Martinho et al [19,21,81,82] and Duhamel et al, [86,87,93] a blob model was proposed for the investigation of polymer chain folding by the fluorescence technique and successfully correlated the temperature dependence of I e /I m of pyrene-labeled chains with their size shrinkage during collapse. (3) It would be excellent to directly follow the time dependence of coil sizes during collapsing.…”