Environmental effects on the protonation states of active site residues in bacteriorhodopsin

Sampogna, Rosemary V.; Honig, Barry

doi:10.1016/s0006-3495(94)80925-7

Cited by 97 publications

(88 citation statements)

References 37 publications

(58 reference statements)

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“…reported to have perturbed shapes (18)(19)(20)(21). We now establish that these shapes can be used as a diagnostic tool to determine the location of the active site.…”

Section: Summary and Discussionmentioning

confidence: 62%

“…This requires calculation of the electrostatic potentials, for which we employ the UHBD (16) program. We use the program HYBRID (15,17) to calculate the mean net charge as a function of pH (18)(19)(20)(21) for each ionizable group (all Lys, Arg, Asp, Glu, His, Tyr, Cys, N terminus, and C terminus) of each protein. For each residue type in a given enzyme, a plot is drawn of predicted mean net charge as a function of pH-THEMATICS.…”

Section: Methodsmentioning

confidence: 99%

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THEMATICS: A simple computational predictor of enzyme function from structure

Ondrechen

Clifton

Ringe

2001

Proc. Natl. Acad. Sci. U.S.A.

215

203

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We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in proteins of known structure. Results are featured for three enzymes: triosephosphate isomerase (TIM), aldose reductase (AR), and phosphomannose isomerase (PMI). We note that TIM and AR have similar structures but catalyze different kinds of reactions, whereas TIM and PMI have different structures but catalyze similar reactions. Analysis of the theoretical microscopic titration curves for all of the ionizable residues of these proteins shows that a small fraction (3-7%) of the curves possess a flat region where the residue is partially protonated over a wide pH range. The preponderance of residues with such perturbed curves occur in the active site. Additional results are given in summary form to show the success of the method for proteins with a variety of different chemistries and structures.T here is a need to develop methods to predict protein function from structure; this need becomes particularly acute as novel protein folds are discovered for which there are no proteins of similar structure with known function. We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in enzymes of known structure. This information will prove to be important in the current challenging quest to predict protein function from sequence and structure.Knowledge of protein sequences and structures has burgeoned very recently as a result of genome sequencing (1) and structural genomics efforts. To translate such information into tangible benefits to humankind, the next step is to develop methods that enable one to predict and to establish function from structure. Techniques used to predict function from sequence or function from structure are in their infancy and rely either on analogies to related proteins of known function (2-8) or on computational searches for binding sites by docking (9) of selected sets of small molecules onto the structure. For instance, recent work has searched the Protein Data Bank (PDB; ref. 10; http:͞͞ www.rcsb.org͞pdb͞) for previously unrecognized cation-binding sites (11).However, there is as yet no reliable method to identify active sites of enzymes or other interaction sites of proteins in the absence of biochemical data, even when the structure is known. There is a wealth of biochemical data that demonstrates that active-site residues involved in specific kinds of chemistry possess predictable chemical properties that enable one to identify them as active-site residues. The most important of these properties is a perturbed pK a that can be determined experimentally by pH titrations of the activity of the enzyme. Herein we demonstrate that theoretical titration functions can identify active-site residues that are involved in Brønsted acid-base chemistry for a variety of proteins, thereby identifying the active site from the location of the residues.In an experimental titration curve, one typically plots pH as a function of the volume of...

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“…reported to have perturbed shapes (18)(19)(20)(21). We now establish that these shapes can be used as a diagnostic tool to determine the location of the active site.…”

Section: Summary and Discussionmentioning

confidence: 62%

Section: Methodsmentioning

confidence: 99%

THEMATICS: A simple computational predictor of enzyme function from structure

Ondrechen

Clifton

Ringe

2001

Proc. Natl. Acad. Sci. U.S.A.

215

203

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“…also in agreement with earlier Poisson equationbased calculations of apparent pK a values in bacteriorhodopsin. 14,34,37 The second-lowest state differs from the lowest only in having a proton transferred from SB to Asp85, as would happen in the M-intermediate. The third and fourth-lowest states differ by having one fewer, and one more protons, respectively, and according to equation (1), the pH dependence is coupled to the total number of protons.…”

Section: Br (Equilibrium) Statementioning

confidence: 99%

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Onufriev

Smondyrev

Bashford

2003

Journal of Molecular Biology

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Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the photocycle, and affinity-switch models, which focus on changes in proton affinity of groups along the transport chain during the photocycle. Using newly available structural data, and adapting current methods of protein protonation-state prediction to the non-equilibrium case, we have calculated the relative free energies of protonation microstates of groups on the transport chain during key conformational states of the photocycle. Proton flow is modeled using accessibility limitations that do not change during the photocycle. The results show that changes in affinity (microstate energy) calculable from the structural models are sufficient to drive unidirectional proton transport without invoking an accessibility switch. Modeling studies for the N state relative to late M suggest that small structural re-arrangements in the cytoplasmic side may be enough to produce the crucial affinity change of Asp96 during N that allows it to participate in the reprotonation of the Schiff base from the cytoplasmic side. Methodologically, the work represents a conceptual advance compared to the usual calculations of pK a using macroscopic electrostatic models. We operate with collective states of protonation involving all key groups, rather than the individualgroup pK a values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for nonequilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems.

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“…In addition, the dielectric constant of the membrane spanning environment is unknown which limits the prediction of whether Asp 804 or Asp 808 exist in a protonated or charged form. In fact, high pK a values have been determined for transmembrane carboxyls not involved in salt bridges or proton transport (in conjunction with a proton-acceptor group) (51). The current understanding of the enzyme structure does not allow us to hypothesize on pK a values for these carboxyls or their possible neutralizing role during cation transport.…”

Section: Structural Functional Status Of Aspmentioning

confidence: 99%

“…In accordance with our data, the essential role of Asp 804 and Asp 808 in cation coordination or transport is supported by the conservation of these amino acids in all species and isoforms of the Na,K-ATPase (4). In addition, Asp 808 is also conserved in other P-type ATPases while Asp 804 is replaced with Asn in the SR Ca-ATPase, Glu in the H,K-ATPase, and is absent in the plasma membrane Ca-ATPase (51,52). Moreover, it must be noted that all of the substituted proteins, with the exception of the conservative substitution, Asp 808 3 Glu, resulted in a similar loss of K ϩ -antagonism of ouabain binding.…”

Section: Structural Functional Status Of Aspmentioning

confidence: 99%