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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 t
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 t
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Attribution Non-Commercial (BY-NC)
Доступные форматы
Скачайте в формате PDF или читайте онлайн в Scribd
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 t
Авторское право:
Attribution Non-Commercial (BY-NC)
Доступные форматы
Скачайте в формате PDF или читайте онлайн в Scribd
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
inic DEGF crystal, Data was collected to 2.0A resolution with a cumulative Rmorge 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 datu reduction procedures.
Difference fouriers were calculated using a refined model (without waters) of hi-MW
bFGF for phases. 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 10c 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 OE2 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 OE1 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-Fe 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. Ttis 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 PtF. 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 OE2 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 keal/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 keal/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 clectronic 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
SCF.