Chapter

Water Compartments in Cens

Gary D. Fullerton* and Ivan L. Cameron'*

Contents

1. Introduction 2

2. The Stoichiometric Hydration Model (SHM) 3

2.1. Water bridges 3

2.2. Methods used to calculate compartmental capacities 3

2.3. Solvent-accessible surface area calculation method 5

2.4. SASA compensation assuming compact folding of globular proteins 8

2.5. SASA estimate for globular proteins based on the hydration model 9

3. Predictions Using the SHM 9

3.1. Prediction of hydration capacities hRa, hB, hPr, and hM(native) 9

3.2. Comparison of hydration model predictions to measured capacities 11

4. Biophysical Measurements of Compartmental Hydration Capacities 11

4.1. Selection of methods 11

4.2. Gravimetric measurement of hydration 12

4.3. Protein rehydration rate method 13

4.4. Differential scanning calorimetry method 15

4.5. Proton NMR titration method 15

4.6. Osmotic compression method 15

5. Relationship of the Hydration Model to Osmosensing and Osmosignaling 18

6. Relationship of the SH Model to Enzyme Function 19

6.1. Enzyme hydration and activity 19

6.2. Neutron scattering assessment of enzyme motion and activity 21

6.3. The SH model predicts both vibrational dynamics and enzyme activity changes 23

* Department of Radiology, University of Texas HSC at San Antonio, San Antonio, Texas ^ Department of Cellular and Structural Biology, University of Texas HSC at San Antonio, San Antonio, Texas

Methods in Enzymology, Volume 428 © 2007 Elsevier Inc.

ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28001-2 All rights reserved.

7. Relationship of the SH Model to Cellular Function 24

7.1. Cellular function depends on water 24

7.2. Hydration compartments and cellular systems correlate with

SH model predictions 24

8. Summary and Conclusions 25 References 25

Abstract

Human experience in the macrobiological world leads scientists to visualize water compartments in cells analogous to the bladder in the human pelvis or ventricles in the brain. While such water-filled cellular compartments likely exist, the volume contributions are insignificant relative to those of biomolecular hydration compartments. The purpose of this chapter is to identify and categorize the molecular water compartments caused by proteins, the primary macro-molecular components of cells. The categorical changes in free energy of water molecules on proteins cause these compartments to play dominant roles in osmoregulation and provide important adjuncts to fundamental understanding of osmosensing and osmosignaling mechanisms. Water compartments possess differences in molecular motion, enthalpy, entropy, freezing point depression, and other properties because of electrostatic interaction of polar water molecules with electric fields generated by covalently bound pairs of opposite charge caused by electronegative oxygen and nitrogen atoms of the protein. Macro-molecules, including polypeptides, polynucleotides, and polysaccharides, are stiff molecular chains with restricted folding capacities due to inclusion of rigid ring structures or double amide bonds in the backbone sequence. This creates ''irreducible spatial charge separation'' between positive and negative partial charges, causing elevated electrostatic energy. In the fully hydrated in vivo state of living cells the high dielectric coefficient of water reduces protein electrostatic free energy by providing polar ''water bridge networks'' between charges, thereby creating four measurably different compartments of bound water with distinct free energy differences.

1. Introduction

This chapter describes general considerations regarding experiments used to detect and measure compartmental hydration capacities of proteins, the primary macromolecular components of cells, and to predict the influence of hydration compartments on cellular osmoregulation using the red blood cell (RBC) as a model cellular system. The discussion embarks with a brief discussion of the electrostatic energy source for directly binding different categories of water molecules as single water bridges, double water bridges, dielectric water clusters (DWC) around the bridges, and finally as encapsulated bulk water sequestered inside the protein volume. The description leads to presentation of molecular calculation methods to predict compartmental water capacities and confirms predictions by comparison to biophysical measurements on fibrillar collagen and two globular proteins, serum albumin and lysozyme. Model predictions are validated by comparison to a wide range of biophysical measurements from the literature to confirm the wide extent of observations attributable to compartmental hydration effects. Four methods used to measure compartmental capacities of single proteins and/or cells accurately are described to assist experimental confirmation of predictions by the reader. The discussion includes a brief review of enzyme behavior as a function of compartmental hydration to facilitate the translation of cellular water and protein hydration compartment concepts to commercial biotechnology applications and concludes by relating molecular hydration mechanisms as source causes of osmosensing, osmosignaling, and osmoregulation effects.

2. The Stoichiometric Hydration Model (SHM)

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