Hydration and protein dynamics: frequency domain fluorescence spectroscopy of proteins in reverse micelles

Pasquina Marzola,Enrico Gratton

doi:10.1021/j100176a083

Abstract

Lysozyme, human serum albumin (HSA), and liver alcohol dehydrogenase (LADH) have been studied in reverse micelles by frequency domain fluorescence spectroscopy. The emission of the tryptophanyl residues of the proteins was monitored. Fluorescence and anisotropy decays were measured from 2 to 350 MHz for each protein in reverse micelles and in aqueous solutions. The wide range of modulation frequencies available allowed direct monitoring of the internal motions of tryptophan residues, occurring in the subnanosecond time range, together with the whole protein rotational dynamics in the micelles. The results indicate that the rotational correlation times for the internal motions and the overall protein rotation in reverse micelles decrease with increasing water concentration. Lysozymes showed peculiar rotational dynamics which reflect denaturation occurring as the protein increases its water content in the reverse micelle. This effect was not observed for the other proteins. Dynamic measurements appear useful in understanding structural changes arising from the interactions between proteins and micellar systems. © 1991 American Chemical Society.

Highlights

The activation of dynamics in proteins is of major importance for their functionality
Recent study on lysozyme hydration indicates that the internal protein dynamics are completely activated at h = 0.15, and that the internal motions are relatively uncoupled from the surface water motion^.^
The Journal of Physical Chemistry, Vol 95, No 23, 1991 9489 nosecond time range.I4*l5 In the present work we focused our attention on three proteins: lysozyme, human serum albumin (HSA), and liver alcohol dehydrogenase (LADH); their fluorescence properties have been characterized in aqueous solution,I6 and two of them have been extensively studied in reverse micellar systems."~"~'*

Summary

Introduction

The activation of dynamics in proteins is of major importance for their functionality. Despite the amount of experimental evidence on protein and the advances in theoretical method^,^^^ there are still some basic aspects that need to be understood Among these aspects is the role of hydration.'" The effect of water is minimal on the protein structure but it is paramount on its dynamics.'-s The effect of hydration on protein dynamics has been addressed very little in the literature, and its physical origin is virtually unknown. A proposed way to elucidate the role of water on protein dynamics is to study the process of adding water to dry proteins up to the solution state. It has been shown that some enzymes, when hosted in reverse micelles, display activity which can be larger than in aqueous solution." In addition, their use is important in carrying out enzymatic ''Address correspondence to this author

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