Proton Travel in Green Fluorescent Protein - Fluorescent Proteins: From Understanding to Design - page 166

Keywords Fluorescence correlation spectroscopy Hydrogen bond network

pH jump Proton antenna QHOP molecular dynamics

Contents
1 Background on Biological Proton Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
2 Fluorescence Autocorrelation of GFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3 pH-Jump Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
4 Hydrogen-Bonded Clusters in GFP X-Ray Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5 Computational Approaches to Study Biomolecular PT Dynamics . . . . . . . . . . . . . . . . . . . . . . . . 177
6 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

1 Background on Biological Proton Transfer

Proton-transfer (PT) reactions are essential parts of many biochemical and bioener-
getic processes [ 1 , 2 ], e.g., in enzymatic reactions [ 3 ] or along hydrogen-bonding
networks through entire membrane proteins such as bacteriorhodopsin or cyto-
chrome c oxidase. PT in hydrogen-bonded networks can either proceed via struc-
tural (Grotthuss-) diffusion or vehicle (Stokes-) diffusion [ 1 ]. In the latter case, the
proton remains bound to one particular diffusing (water) molecule, whereas in the
former case it changes continuously its partner by breaking and reforming covalent
bonds [ 4 ]. In proteins, the thermodynamic and kinetic properties of PT processes
are strongly influenced by electrostatic interactions with the environment of the
proton-donating and -accepting chemical groups [ 5 , 6 ]. By affecting both the
relative energetic difference between the reactant and product states and the barrier
height, environmental effects are of crucial importance for reaction rates, equilibria,
and local p K a values of titratable residues. Things may be complicated further
because (1) proton- and electron-transfer processes are often coupled as, for
example, in cytochrome c oxidase, (2) proton transport across entire proteins
involves a large number of subsequent PT events, and (3) proton migration often
involves concerted transfers and quantum-mechanical tunneling.
For many biological systems involved in proton pumping, such as the bacterial
photosynthetic reaction center and cytochrome c oxidase, the biomolecular rate
constant for proton uptake from bulk solution was found to significantly exceed the
rate for proton diffusion through water to a single surface-bound titratable group
[ 7 ]. Gutman and Nachliel gave a possible explanation for this behavior by propos-
ing the so-called antenna effect [ 8 ]. There, they suggested that such rapid proton-
transfer reactions indicate the involvement of multiple negatively charged Asp and
Glu residues on the protein surface that are titratable and can capture protons from
bulk solution. The closely located charged residues might thus extend the proton
capture area of the surface and provide a local, two-dimensional buffer composed
of rapidly proton-exchanging titratable sites.
Providing definite experimental evidence for proton-transfer phenomena in
biomolecules is quite hard. One basic technique is to perform time-resolved