Protein titratable groups

In molecular mechanics and molecular dynamics studies of proteins, assig-ment of standard, non-dynamical ionization states of protein titratable groups is a common practice. This assumption seems to be well justified because proton exchange times between protein and solution usually far exceed the time range of the MD simulations. We investigated to what extent the assumed protonation state of a protein influences its molecular dynamics trajectory, and how often our titration algorithm predicted ionization states identical to those imposed on the groups, when applied to a set of structures derived from a molecular dynamics trajectory [34]. As a model we took the bovine... 

The procedure is computationally efficient. For example, for the catalytic subunit of the mammalian cAMP-dependent protein kinase and its inhibitor, with 370 residues and 131 titratable groups, an entire calculation requires 10 hours on an SGI 02 workstation with a 175 MHz MIPS RIOOOO processor. The bulk of the computer time is spent on the FDPB calculations. The speed of the procedure is important, because it makes it possible to collect results on many systems and with many different sets of parameters in a reasonable amount of time. Thus, improvements to the method can be made based on a broad sampling of systems. 

Continuum electrostatic approaches based on the Poisson equation have been used to address a wide variety of problems in biology. One particularly useful application is in the determination of the protonation state of titratable groups in proteins [46]. For... 

One way in which to probe the structural surroundings of a protein is to monitor the pH behavior of specific carbon sites of the C probes. pH-titration studies, of given resonances, had previously been used for probing of the protein structure, because they are known to provide information concerning electrostatic (salt-bridging) interactions in the protein, neighboring group-ionizations, and local environments. ... 

With multiple ionizable groups, such as in amino acids and proteins, each group titrates separately according to its pKa. The titration curves shown in Fig. 23-5 are for the amino acids glycine, histidine, and glutamate. 

Among protein aromatic groups, histidyl residues are the most metal reactive, followed by tryptophan, tyrosine, and phenylalanine.1 Copper is the most reactive metal, followed in order by nickel, cobalt, and zinc. These interactions are typically strongest in the pH range of 7.5 to 8.5, coincident with the titration of histidine. Because histidine is essentially uncharged at alkaline pH, complex-ation makes affected proteins more electropositive. Because of the alkaline optima for these interactions, their effects are most often observed on anion exchangers, where complexed forms tend to be retained more weakly than native protein. The effect may be substantial or it may be small, but even small differences may erode resolution enough to limit the usefulness of an assay. 

Ribonuclease is an enzyme with 124 amino acids. Its function is to cleave ribonucleic acid (RNA) into small fragments. A solution containing pure protein, with no other ions present except H+ and OH- derived from the protein and water, is said to be isoionic. From this point near pH 9.6 in the graph, the protein can be titrated with acid or base. Of the 124 amino acids, 16 can be protonated by acid and 20 can lose protons to added base. From the shape of the titration curve, it is possible to deduce the approximate pATa for each titratable group.1-2 This information provides insight into the environment of that amino acid in the protein. In ribonuclease, three tyrosine residues have "normal values of pATa(=10) (Table 10-1) and three others have pA a >12. The interpretation is that three tyrosine groups are accessible to OH, and three are buried inside the protein where they cannot be easily titrated. The solid line in the illustration is calculated from pA"a values for all titratable groups. 

Many amine bases and carboxylic acids in proteins titrate with anomalously high or low pA s (Table 5.4). The reasons are quite straightforward, and depend on the microenvironment. If a carboxyl group is in a region of relatively low polarity, its pKa will be raised, since the anionic form is destabilized. Alternatively, if the carboxylate ion forms a salt bridge with an ammonium ion, it will be stabi-... 

The dissociation of titratable groups of a protein molecule is influenced by the electrolyte concentration. A positively charged protein will contain more bound protons per molecule at higher electrolyte concentrations, and a negatively charged protein will contain fewer bound protons per molecule at higher concentrations. 

Fig. 1. Formulas for the most important titratable groups of protein molecules. The model compounds listed in Table I were chosen to resemble those groups as closely as possible.

The relation between structure and acidity of organic compounds has been the subject of much study. Those aspects which are of interest in connection with protein titration curves have been reviewed in definitive manner by Edsall and Wyman (1958) and by Edsall (1943), and the reader is referred to these reviews for a discussion of the theoretical and empirical principles which are involved. For the present purpose it is sufficient to extract the data which will lead to the expected pK values of the titratable groups of proteins, and this has been done in Table I. 

Conclusion. If protein molecules exhibit no interactions that are not also present on smaller molecules, then the pK values of their titratable groups would be expected to be roughly those of Table I. Electrostatic forces may move them up or down by as much as 1.5 pK units, but relative values will be unaffected thereby. The pK changes during the course of titration, so that the titration curve for any one group will be broader than it would be for a monobasic acid. 

In terms of the expected pK values of Table I, the acid end point is the point where all titratable groups listed there, except the phosphate group, are expected to have been converted to their acidic forms. (Considering that most proteins have a large positive charge at the acid end point, the expected pKi for the phosphate group would be zero or less.)... 

Spectroscopic methods for following the titration of other common titratable groups of protein molecules do not exist. The reason is that the peptide group and the aromatic rings of phenylalanine, tryptophan, and tyrosine side chains absorb strongly in the ultraviolet below 250 m/i, making it essentially impossible to observe the relatively small changes in absorb-... 

Counting of titratable groups is particularly interesting when the titration curve for a protein depends on the conditions under which it is determined, as for instance in the example of Fig. 2, where the titration curve above pH 9.7 depends on time. What is the physical meaning of the... 

A much more revealing treatment is one which specifically takes into account the fact that many of the titratable groups of a protein molecule are likely to be intrinsically identical, or very nearly so. Furthermore, it takes into account the one interaction between titratable groups which is... 

We now introduce the second important feature of the Linderstrpm-Lang treatment, that many of the titratable groups of any protein molecule are intrinsically identical. In other words, we can divide the titratable... 

If Ml is the true molecular weight of the protein, Wi the corresponding value of the electrostatic interaction factor, and Zi the corresponding charge at any pH, then, for any titratable group with given pKint,... 

Table V shows that the vast majority of the titratable groups of the smaller protein molecules have pK nt values which are quite close to the values predicted from the pK s of model compounds. This feature of protein titration curves has been well known for a long time, and is accepted as normal. It is however really an astonishing result, for it implies that most of the titratable groups of the smaller protein molecules are in as intimate contact with the solvent as similar groups on smaller molecules, and that they are able to accept or release hydrogen ions in this location without requiring any modification of the protein conformation in the vicinity of the titratable group. Since most of the proteins examined have been globular proteins, tightly folded so as to exclude solvent from most of the interior portions, the titratable groups must be nearly always at the surface.

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