Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding,☆☆

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Highlights

  • Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding.

  • Volume changes upon substitutions at buried sites depend on structural plasticity of proteins.

  • Smaller proteins have positive volume changes upon unfolding.

Abstract

Background

Pressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules.

Scope of this review

This review provides novel details of the use of PPC to analyze unfolding transitions in proteins. The emphasis is made on the data analysis as well as on the validation of different structural factors that define the volume changes upon unfolding. Four case studies are presented that show the application of these concepts to various protein systems.

Major conclusions

The major conclusions are:

  • 1.

    Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles.

  • 2.

    The changes in the thermal expansion coefficient upon unfolding appear to be temperature dependent.

  • 3.

    Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding.

  • 4.

    Structural plasticity of proteins defines the position dependent effect of amino acid substitutions of the residues buried in the native state.

  • 5.

    Small proteins have positive volume changes upon unfolding which suggests difference in balance between the cavity/void volume in the native state and the hydration volume changes upon unfolding as compared to the large proteins that have negative volume changes.

General significance

The information provided here gives a better understanding and deeper insight into the role played by various factors in defining the volume changes upon protein unfolding. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Introduction

How a linear sequence of amino acids folds into an intricate and biologically active three-dimensional structure of a protein has captivated the scientific community ever since the myoglobin structure was reported [1], [2]. Multiple physico-chemical forces such as hydrogen bond, hydrophobic interaction, van der Waals interaction, disulfide bridges and electrostatic interactions have been widely discussed for their role in defining the protein stability (see e.g. [3], [4], [5], [6], [7]. Much of the studies have been exploring the effect of fundamental environmental parameter, temperature (T) on protein stability (defined as the Gibbs energy difference between unfolded and native states ΔG = GU  GN). However, much less has been done to explore the dependence of the stability of proteins on the other equally fundamental environmental parameter, hydrostatic pressure (P). This is important to understand the physico-chemical principles that define the stability of proteins in general and in particular for the organisms that live in the deep sea, the so-called barophiles [8], [9].

The pressure dependence of the protein stability is defined by the volume changes upon unfolding, ΔV:ΔV=VUVN=ΔGPT

where VU and VN are the volumes of the unfolded and native states, respectively. If ΔV is negative, increase in hydrostatic pressure will, according to Le Châtelier's principle, lead to a decrease in protein stability, while positive values of ΔV will lead to an increase in stability. There are two major factors contributing to the ΔV of protein unfolding. The first factor is the imperfection in the packing of the native proteins, i.e. well documented presence of cavities of voids [10], [11], [12]. The second factor is the volume changes of solvent water upon hydrating the protein groups exposed due to the unfolding [13]:ΔV=Vvoids+VHydration.

The exact magnitudes of these effects, especially the hydration of newly exposed accessible surface area due denaturation, are still under debate [14], [15], [16].

Until recently, the volume changes upon unfolding have been determined indirectly from the pressure dependence of equilibrium constant (see e.g. ref.[16]). Introduction of the commercial instruments to perform pressure perturbation calorimetry (PPC) experiments [17] and subsequent development of data analysis formalism [18] provided the protein folding community the ability to directly determine ΔV of conformational transitions. These measurements combined with other biophysical methods assessing thermodynamic (e.g. DSC) and structural (e.g. circular dichroism and fluorescence spectroscopies, NMR, SAXS) properties of proteins and supported by computational modeling allowed better understanding of the properties of proteins that contribute to the pressure dependence of protein stability [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40].

In this paper we report several robust approaches to analyze the PPC profiles of proteins and report four novel case studies of protein systems that clarify the importance of various factors in defining the volume changes upon protein unfolding. The article is organized as follows. After the Materials and methods section, we present a brief discussion of the PPC experiment and foundation for a two-state analysis. This is followed by case studies of four different protein systems for which PPC experiments were performed. Each case highlights certain aspects of data analysis and more importantly, probes various structural aspects of proteins that define their volume changes upon unfolding.

Section snippets

TrpZip peptide

TRPZIP4 with sequence GEWTWDDATKTWTWTE-NH2 was synthesized using standard Fmoc chemistry as described previously [41]. The peptide has a molecular weight of 2013 Da and extinction coefficient (ε280,0.1%) of 11.41 optical units. The partial specific volume, V̅pr, was calculated to be 0.70 cm3/g based on the amino acid composition as described previously [42]. Both DSC and PPC experimental procedures were performed using TRPZIP4 concentrations between 0.5 and 3.5 mg/ml. Peptide purification was done

Experimental determination of thermal expansion coefficient of proteins and volume changes upon unfolding

In a PPC experiment, the thermal expansion coefficient is determined from the heat effects ΔQbuf/pr(T), produced in the sample cell (containing protein in a buffer) relative to the reference cell (that has that same buffer alone) as both cells are subjected to rapid and small amount of perturbations in hydrostatic pressure (ΔP ~ 5 atm) under isothermal conditions [17]. The heat effects, ΔQbuf/pr(T), are proportional to the thermal expansion coefficient as:αT=αH2OTΔQH2O/bufTTΔPvcellΔQbuf/prTT

Concluding remarks

The four case studies discussed above provide novel details concerning the use of PPC for the analysis of conformational transitions in proteins and also shed more light on factors defining the volumetric properties of proteins.

  • 1.

    Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles. This is particularly important for the transitions that are broad (i.e. low enthalpy) and also for the transitions that have small volume changes.

  • 2.

    The

Acknowledgments

This work was supported by the grants from the US National Science Foundation (CHE-1145407 and CHE-1506468).

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    Note: The authors declare no competing financial interest.

    ☆☆

    This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

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