Tài liệu Báo cáo khóa học: Numerical calculations of the pH of maximal protein stability pptx

Numerical calculations of the pH of maximal protein stability

The effect of the sequence composition and three-dimensional structure

Emil Alexov

Howard Hughes Medical Institute and Columbia University, Biochemistry Department, New York, USA

A large number of proteins, found experimentally to have

different optimum pH of maximal stability, were studied to

reveal the basic principles of their preferenence for a par￾ticular pH. The pH-dependent free energy of folding was

modeled numerically as a function of pH as well as the net

charge of the protein. The optimum pH was determined in

the numerical calculations as the pH of the minimum free

energy of folding. The experimental data for the pH of

maximal stability (experimental optimum pH) was repro￾ducible (rmsd ¼ 0.73). It was shown that the optimum pH

results from two factors – amino acid composition and the

organization of the titratable groups with the 3D structure.

It was demonstrated that the optimum pH and isoelectric

point could be quite different. In many cases, the optimum

pH was found at a pH corresponding to a large net charge of

the protein. At the same time, there was a tendency for

proteins having acidic optimum pHs to have a base/acid

ratio smaller than one and vice versa. The correlation

between the optimum pH and base/acid ratio is significant if

only buried groups are taken into account. It was shown that

a protein that provides a favorable electrostatic environment

for acids and disfavors the bases tends to have high optimum

pH and vice versa.

Keywords: electrostatics; pH stability; pKa; optimum pH.

The concentration of hydrogen ions (pH) is an important

factor that affects protein function and stability in different

locations in the cell and in the body [1]. Physiological pH

varies in different organs in human body: the pH in the

digestive tract ranges from 1.5 to 7.0, in the kidney it ranges

from 4.5 to 8.0, and body liquids have a pH of 7.2–7.4 [2]. It

was shown that the interstitial fluid of solid tumors have

pH ¼ 6.5–6.8, which differs from the physiological pH of

normal tissue and thus can be used for the design of pH

selective drugs [3].

The structure and function of most macromolecules are

influenced by pH, and most proteins operate optimally at a

particular pH (optimum pH) [4]. On the basis of indirect

measurements, it has been found that the intracellular pH

usually ranges between 4.5 and 7.4 in different cells [5]. The

organelles’ pH affects protein function and variation of pH

away from normal could be responsible for drug resistance

[6]. Lysosomal enzymes function best at the low pH of 5

found in lysosomes, whereas cytosolic enzymes function

best at the close to neutral pH of 7.2 [1].

Experimental studies of pH-dependent properties [7–11]

such as stability, solubility and activity, provide the benchmarks

for numerical simulation. Experiments revealed that altho￾ugh the net charge of ribonuclease Sa does affect the

solubility, it does not affect the pH of maximal stability or

activity [12]. Another experimental technique as acidic or

basic denaturation [13–15] demonstrates the importance of

electrostatic interactions on protein stability.

pH-dependent phenomena have been extensively mode￾led using numerical approaches [16–19]. A typical task is to

compute the pKas of ionizable groups [20–26], the isoelectric

point [27,28] or the electrostatic potential distribution

around the active site [29]. It was shown that activity of

nine lipases correlates with the pH dependence of the

electrostatic potential mapped on the molecular surface of

the molecules [29]. pH dependence of unfolding energy was

modeled extensively and the models reproduced reasonable

the experimental denaturation free energy as a function of

pH [19,30–36].

The success of the numerical protocol to compute the

pH dependence of the free energy depends on the model

of the unfolded state, the model of folded state and thus

on the calculated pKas. It is well recognized that the

unfolded state is compact and native-like, but the magni￾tude of the residual pairwise interactions and the desol￾vation energies has been debated. Some of the studies

found that any residual structure of the unfolded state has

negligible effect on the calculated pH dependence of

unfolding free energy [31], while others found the opposite

[33–36]. It was estimated that the pKas of the acidic

groups in unfolded state are shifted by – 0.3 pK units in

respect to the pKas of model compounds. Although

including the measured and simulated pK shifts into the

model of unfolded state changes the pH dependence of

the unfolding free energy, it most of the cases it does not

change the pH of maximal stability [33–36]. Much more

Correspondence to E. Alexov, Howard Hughes Medical Institute and

Columbia University, Biochemistry Department, 630W 168 Street,

New York, NY 10032, USA.

Fax: + 1 212 305 6926, Tel.: + 1 212 305 0265,

E-mail: [email protected]

Abbreviations: MCCE, multi-conformation continuum electrostatic;

SAS, solvent accessible surface.

(Received 15 September 2003, accepted 11 November 2003)

Eur. J. Biochem. 271, 173–185 (2004)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03917.x