High-affinity electrostatic binding of the

noble gas krypton to bFGF:

implications & possibilities
Ogan Gurel
30 November 1993

Experimental

Krypton was introduced at approximately 5 atm into a quartz capillary containing a single triclinic bFGF
crystal. Data was collected to 2.0A resolution with a cumulative Rmerge of 3.3% and an overall
completeness of 80%. Native data was also collected (78% complete, 8% Rmerge to 2.0A resolution)
under identical crystallization, data collection and data reduction procedures.

Difference fouriers were calculated using a refined model (without waters) of hi-MW bFGF for phases. A
high-resolution (2A) difference fourier map showed a single strong peak at a height of 356 located near
the surface of the protein in the vicinity of residues 77 and 78. Closely flanking this peak are two smaller,
distinct side peaks at 106 disposed such that all three peaks lie along a line perpendicular to the surface of
the protein between the side chains of glutamate 78 and lysine 77.

Structure of the binding site

The krypton atom is closely packed between the positively charged NZ of lysine 77 and the negatively
charged 0E2 of glutamate 78 at a distance of 4.5 A between atomic centers. The fact that this peak lies
between a positive and a negative charge is strong evidence against a "conventional" anion or cation in the
site. Additional coordination appears to be provided by the positively charged NH2 of arginine 81 and the
negatively charged 0E1 of glutamate 91 but these are at longer distances of 5.8 and 4.9 A respectively.

There is no observable water at the krypton site both in native Fo-Fc maps as well as in other published FGF
studies. There is, however, a high B-value water located somewhat below the site close to glutamates 77 and
91. The Fo-Fo map shows a distinct negative peak overlying the water site indicating that it might have
been pushed out during the binding of krypton although this appears not to be obligatory in steric terms.

We had originally anticipated that the binding site would be hydrophobic in nature. The result we have here of
an almost purely electrostatic interaction is both unexpected and unprecedented.
Noble gas chemistry

Very few noble gas compounds or complexes are known and those that are have been isolated using
powerful reagents and under extreme conditions of temperature and pressure. The krypton site here is
completely stable at room temperature without the benefit of any extraordinary pressures. The existence of
such a complex speaks strongly about the almost incredible specificity and affinity of binding between
proteins and their ligands. It is all the more amazing when one considers that krypton is spherically
symmetric and uncharged -- characteristics that don't immediately lend themselves to any special specificity.

Most noble gas chemistry involves the oxidation of xenon (or more rarely, krypton) by powerful oxidizing
agents such as PtF6. In addition, the noble gases with their electron rich valence shells have been
classified as extremely weak Lewis bases. In chemical terms, the krypton-FGF coordination follows
the latter pattern with electrons being "donated" to the electrophilic proton on NZ of lysine 77 which is
facilitated by the polarizing effect of 0E2 on glutamate 78.

Thermodynamics of binding

A very rough calculation of the enthalpy of condensation and the entropy of a monatomic gas indicate that the
minimum free energy of binding must be -11 kcal/mol. Additional binding energy must also be devoted to
polarizing the krypton and to maintaining the site at nearly 100% occupancy This leads to free energies of
binding that are on the order of -15 to -20 kcal/mol. All of these are very gross numbers and need to be
refined with a better understanding of the structure of the site. Nevertheless, it is clear that the binding of
the protein to this symmetric, uncharged atom is both strong and specific. The krypton site represents a
unique opportunity to elucidate the nature of strong, specific binding at the single atom level.

Electrostatic considerations

In terms of ligand binding the most important issue is the strength of the electric field at the krypton site.
This will ultimately determine both the electronic polarization of krypton as well as the overall energy of
binding to the resultant dipole. Current paradigm separates protein electrostatics into two regimes: an inner,
low dielectric regime, and an outer, high dielectric regime. However, in view of the required electric field
strength and the absence of water in the krypton binding site, it appears that the dielectric constant might
be quite low at this point and perhaps at other critical regions on the surface of the protein. In other words,
the "two dielectric" regime would not have predicted the possibility of such a binding site.

Additionally, most models of electrostatically mediated ligand binding assume essentially nonpolarizable
atoms. Again, under these assumptions, the krypton binding site would also not have been predicted. This
example, then, indicates that current electrostatic formulations need to reevaluate the role of electronic
polarization.
Ligand-binding: induced fit on an electronic scale

The current paradigm of protein-ligand binding assumes an "inverse" Born-Oppenheimer approximation. In

other words, it is the nuclei that move in response to forces, while the electrons are essentially stationary.
Primitive "docking" studies exemplify this approach. Again, without an appreciation of electronic
polarization effects, such a krypton binding site would not have been predicted. Ligand-binding theories
clearly have to take into account dynamic effects at the level of the electrons in addition to the gross
movements of atoms.

One further consequence is that polarization in general may be an important factor in ligand binding. In
other words, just as we've seen the importance of electronic polarization -- atomic and orientational
polarizations may also be relevant, if not critical, to the binding process.

Theories of anesthetic action

Current theories of anesthetic action hold that small anesthetics such as xenon bind predominantly to
hydrophobic sites, while larger anesthetics bind to a combination of hydrophobic and polar sites. By
implication, it has been thought that the greater efficacy of these large anesthetics is due to their polar
mode of binding which presumably interferences with hydrogen bonding patterns in various cellular
substructures. The evidence for an electrostatic binding site for krypton, however, puts these theories into
question.

Protein Stability

Proteins are stabilized by the folding of their hydrophobic regions into the core of the molecule and the
consequent presentation of charged surfaces to the exterior aqueous solvent. However, there is evidence
that some very highly charged proteins can in fact be relatively unstable. A possible explanation may be the
existence of surface pockets of very high electric field intensity. Current paradigm has focused on
manipulating the interior of proteins in order to influence stability -- witness the innumerable lysozyme
mutants. However, mutation of critical surface charges may also significantly affect protein stability.

Potential phasing power

As discussed before, krypton can serve as an effective anomalous phasing vehicle (physically -- though
not chemically -- analogous to selenomethionine). Preparation of the derivative is relatively easy and it
appears that highly specific, low B-value, high occupancy sites may be acheivable in a relatively general
fashion. Experiments are now in progress to use krypton to provide phase information in the structure
determination of S CF.