WHAT MAKES PROTEINS WORK: Exploring Life in P-T-X

Abstract

Although considerable progress has been made in the molecular biophysics of proteins, it is still not possible to reliably design an enzyme for a given function. The current understanding of enzyme function is that both structure and flexibility are important. Much attention has been focused recently on protein folding and thus structure, spurred on by insights from the folding funnel concept. For experimental studies of protein folding, variations in temperature (T) and chemical composition (X) of the solution have been traditionally exploited, although more recent studies using variations in pressure (P) made possible through new instrumentation have led to a deeper understanding of the energy landscape of protein folding. Other work has shown that flexibility is also essential for enzymes, although it is still not clear what type is important. Another avenue has been to take advantage of “Nature’s laboratory” by exploring homologous proteins from organisms that live in extreme conditions, or “extremophiles”. While the most studied extremophiles live at extremes of T and X, recent exploration of deep-sea environments has led to the discovery of organisms living under high P, or “piezophiles”. An exploration of targeted enzymes from organisms with various P-T-X growth conditions coupled with advances in biophysical instrumentation and computer simulations that allow studies of these enzymes at different P-T-X conditions may lead to a better understanding of “flexibility” and to general design criteria for active enzymes.

Preface

Kamal Shukla’s great contribution to science has been his vision that physical sciences could bring new insights to biological sciences, and that the marriage of methodologies, particularly theoretical/computational with experimental, was needed to tackle the complexities of biology. Furthermore, his openness to new methods and different ideas outside the current fad has helped make his vision a reality. In my remarks below, I have not tried to limit myself to projects that I know Kamal had sponsored, nor have I tried to highlight all that he has sponsored. Instead, everything I mention has been influenced directly or indirectly by his efforts. Perhaps the indirect influences are most telling, because they would not have happened without Kamal.

INTRODUCTION

While we are beginning to understand how many individual proteins work, we still do not really understand what makes proteins in general work. The physical insights from the concept of the protein-folding funnel have driven advances in understanding protein folding and stability over the past 25 years, as discussed more thoroughly elsewhere in this issue. In the same way, advances in fundamental concepts for what characteristics make proteins able to function are needed. Although the need for structure and flexibility has long been recognized, characterization of the flexibility is still unclear. The recent exploration for organisms that live in extreme conditions and new instrumentation for biophysical studies leads to the opportunity for understanding how the macromolecules that comprise these organisms are able to function under these extremes. In particular, exploiting the ability to alter pressure as well as temperature and chemical composition may lead to a better characterization of flexibility. Obvious implications are for designing functional proteins for biomedicine and biotechnology. Subtler implications are for understanding the origins of life and evolution, as well as for determining the “limits of life” for guiding the search for life both terrestrially and extra-terrestrially. The focus here is on what makes functional enzymes, which are requirements for growing organisms.

PAST ACCOMPLISHMENTS

Biophysical studies of the structure and dynamics of proteins

Biophysical studies have led to two concepts have guided our ideas for protein function for many decades. By the early 1970’s, X-ray crystal structures of proteins showed that the structure is important, and helped introduce the idea that conformational flexibility is also important since structures were observed to have different conformations that were thought to represent different stages of activity. However, the transitions between these conformations were viewed as “rigid body” motion of domains, “Kabuki proteins” (to borrow a phrase from Benoit Roux) that jumped from pose to pose.

A revolution in our thinking of how proteins work came in the late 1970’s, when the first molecular dynamics computer simulations [1] and a variety of experiments including Nuclear Magnetic Resonance (NMR) spectroscopy [2] and X-ray crystallography [3] demonstrated that individual atoms in proteins exhibit significant thermal fluctuations, i.e., the “jigglings and wigglings of atoms” [4]. Rather than Kabuki proteins, proteins begin to be viewed as fluid-like and deformable. Within the next ten years, atomic fluctuations were shown to be vital in protein function, not only in making transient pathways for substrates to active sites (Fig. 1) but also in allowing conformational changes to occur smoothly using collective motions [5]. Exploration of the ‘glass transition’ in protein led to further characterization of the energy landscape of folded proteins as individual atomic vibrations below the transition and as collective modes superimposed on the atomic vibrations above the transition [6].

Figure 1.

Early example of a molecular dynamics simulation that demonstrated the importance of atomic fluctuations in the biological function of a protein: trajectory of a CO molecule escaping from myoglobin. Figure from ref. [5]

Since then, much progress has been made in determining how individual proteins function at an atomic level, including enzymes, membrane channels and pores, etc. This progress was made possible by numerous X-ray and NMR structures that have been solved and deposited in the Protein Data Bank (PDB) [7]. Computational methods proved critical in understanding the energetics as well as in providing missing links that could not be captured by structural methods. In addition, site-specific mutagenesis provided a powerful tool to test hypotheses about the role of specific residues or a few key residues.

Extremophiles and Nature’s laboratory

An addition to our toolbox of methods for studying proteins came in the late 1980’s with the discovery of organisms that live in extreme conditions of pressure (P), temperature (T) or chemical composition (X), or “extremophiles”. Studying proteins from extremophiles takes advantage of “Nature’s laboratory”, which has generated random changes over eons of time and measured their success by the survival of the fittest organisms under those conditions, or in other words, evolution. Most of work up to now has been on organisms that live in extremes of temperature, both high (thermophiles) and low (psychrophiles). Comparisons of proteins from thermophiles with those from mesophiles indicate that Nature uses a variety of mechanisms to stabilize proteins against high temperature, including more salt-bridges, hydrogen bonds, and van der Waals contacts [8]. Although studies indicate the importance of electrostatics [9], a “unifying set of rules” for thermostability [10] is still elusive [8]. However, the type of mechanisms used in the proteins of an organism might have implications in understanding its evolution. For instance, the observation that proteins from thermophilic archaea are generally more compact while those from thermophilic bacteria generally have a few strong interactions indicates mesophilic archaea may have “de-evolved” from primordial thermophilic archaea in hot environments by slowly losing many weak interactions while thermophilic bacteria may have evolved from mesophilic bacteria that recolonized a hot environment by adding a few strong interactions in each protein [11].

Since extremophiles actually thrive under extreme conditions, many studies have focused on how their enzymes are able to function under those conditions since enzyme activity is a requirement for growth. A “corresponding states” idea emerged that the maximum activity of homologous enzymes [12] from different organisms was near the growth temperatures of each organism (Fig. 2a). Focusing on monomeric enzymes, both protein stability and flexibility appear necessary to maintain activity [13] since enzymes from thermophiles need to be thermostable while enzymes from psychrophiles need to be flexible at their low growth temperatures. Thus, thermophile enzymes were expected to be more stable and rigid at standard temperatures while psychropile enzymes might actually be more subject to cold-unfolding because of lower stability needed for flexibility (Fig. 2b), just as long as the cold-unfolding temperature is beneath the organism’s growth temperature. A further wrinkle is that in proteins from hyperthermophiles, stability may due to flexibility/mobility that increases conformational entropy rather than rigidity due to extra stabilizing interactions that decreases enthalpy [9], contrary to earlier assumptions mentioned above. However, the answers for thermophiles may differ from hyperthermophiles and different proteins may adopt different solutions. Recent studies at a proteome level [14, 15] may help to resolve this issue.

Figure 2.

Corresponding states and the role of stability and flexibility in enzyme activity: comparison of (a) percentage activity and (b) Gibbs free energy of unfolding (ΔG) as a function of temperature for a homologous enzyme from a psychrophile (blue), a mesophile (black), and a thermophile (red). Figure from ref. [13].

FUTURE CHALLENGES

Much progress has been made in understanding proteins at a molecular level using a combination of experimental, computational, and theoretical biophysical methods. However, while both protein stability and flexibility have been identified as essential for active enzymes, a big question still remains – what does “flexibility” mean? Is it the large conformational changes, or can it be reduced more fundamentally to the magnitude of the atomic fluctuations that give rise to the deformability of a protein, which then influences the large conformation changes? Is it localized to the active site region or is it a global property of the entire protein? Is there even a general type (or types) of flexibility that is necessary for enzyme activity? So far, while there are many clues from studies of enzymes from thermophiles and psychrophiles at different growth temperatures, a clear picture of the nature of flexibility necessary for enzyme activity has not emerged. Moreover, while the approach outlined here is in terms of enzymes, it can be used for other proteins, nucleic acids, and biomembranes, which are also affected by P-T-X. In particular, recent work on the pressure effects on these molecules, as reviewed in [16], makes such studies possible.

Expanding the exploration of Nature’s laboratory

The exploration of Nature’s laboratory can now be expanded to enzymes from microbes that live at high pressures, or “piezophiles”, so a new P-T-X approach to understanding enzymes can be undertaken [17]. Bioinformatics-type searches of Nature’s laboratory could be made for targeted enzymes from organisms adapted to various P-T-X conditions. Then, a variety of experimental and computational methods, including structure determination, thermodynamic measurements, and biophysical/biochemical characterization, could be used to study these enzymes also under different P-T-X conditions. Understanding how enzymes from organisms with different growth conditions respond to different conditions will lead to a better fundamental understanding of enzymes as well as design criteria. In particular, one can begin to construct energy landscapes for active enzymes (Fig. 3) to better understand the physical nature of the flexibility. Comparisons of archaea, bacteria, and eukarya also may lead to discovering more solutions for adaptation if Nature has found different solutions for each domain.

Figure 3.

Building landscapes for enzyme activity: schematics of an “average” atom in an enzyme from a mesophile (m) with that from a homologous enzyme from a piezophile (p) at growth temperature (T) and pressure (P) of mesophile on left and piezophile on right. The force constant k_i, free energy difference between wells ΔG_i, and transition barrier ΔG^‡_i, i = m or p, are indicated for the mesophile at its growth T and P. Left well corresponds to the more favored conformation and right well corresponds to a more open conformation with lower probability.

Why pressure?

The effects of pressure P on mesophile proteins include compression, subunit dissociation, and unfolding, although pressure unfolding [18] tends to occur at higher pressures than where microbial communities have been found [16]. Exploring the effects of high pressure on monomeric enzyme function where subunit dissociation is not relevant is particularly intriguing because of the analogies to low temperature in that compression might reduce fluctuations. Much like psychrophile enzymes, enzymes from piezophiles might be adapted more for flexibility than stability, since many deep sea enzymes appear to be marginally stable and thus non-intuitively unfold at lower pressures compared to their mesophilic counterparts [19]. However, the effects of high pressure on folded proteins are different from those of temperature; for instance, pressure affects the populations of conformational substates of enzymes so that more open, solvated conformations are preferred [20]. Thus, high pressure may actually result in more conformational flexibility. Further confusing the issue is that most piezophiles were isolated from either cold deep-sea environments or near deep-sea hydrothermal vents so adaptations for pressure are difficult to separate from those for temperature. Interestingly, of the piezophiles isolated so far, bacteria have been psychrophilic while archaea have been thermophilic [21].

Why chemical composition? Proteome evolution vs. milieu adaptation

While extremophiles are known to adapt to extreme conditions via adaptations of their proteins, they can also adapt by changing the intracellular milieu. Thus, the definition of chemical composition X for studies of extremophile proteins should be extended beyond typical denaturants to what is found in the intracellular environment. Microbes respond to stresses in their environment by regulating heat and cold shock proteins, organic solutes or “osmolytes”, and inorganic solutes. However, producing shock proteins requires expenditure of considerable cellular energy and resources so that accumulating small co-solutes might be a more efficient permanent adaptation. In particular, pressure-induced osmolytes, or “piezolytes”, have been found [21] but their effects on proteins at different pressures are not clear.

What are implications?

Understanding the nature of flexibility for enzyme function is essential not only for basic knowledge, but has many important implications. The most obvious is reliable bioengineering of enzymes for medical or other purposes including high pressure processing of foods and beverages, or “pascalization”. For these purposes, a “material science” approach to enzymes may be fruitful, so that an understanding is developed of the mechanical properties necessary for enzyme activity and how to measure them so that design criteria can be made. Also, determining how different microbes adapt their enzymes for different conditions has implications for understanding evolution of microbes. In addition, the ability to predict the limits of enzyme function in P-T-X is a step in defining the limiting P-T-X conditions under which microbes can thrive, which could guide the search for life, both in new terrestrial domains as well as in extraterrestrial environments. Closer to our everyday experience, the increasing recognition of the importance of the microbial world to human health and the biosphere leads to concerns about the consequences of the migration of extremophiles into our ecosystem due to upheavals induced by geological hazards, such as arising from global warming.

Why now?

Expanding studies of enzyme flexibility from variations in T-X to variations in P-T-X is particularly timely because of advances in the past few years. Microbes have been found in ever more extreme temperature environments as well as under multiple extremes [22]. Piezophiles have been found that thrive in deep-sea, deep-sea hydrothermal vents, oceanic crust, and continental crust under remarkable extremes of pressure up to 1.3 kbar [16], which would crush their mesophilic counterparts. For instance, recent exploration have found piezophiles from as far down as the bottom of the Challenger Deep in the Marinas Trench [23]. In addition, microbiologists are actively seeking new microbes in extreme environments for a global genome census (https://deepcarbon.net//content/deep-life). Furthermore, improvements in sampling, culturing, and genetic engineering methods [21] means that more enzymes from different microbes will be accessible for a P-T-X approach.

Another timely advance has been in biophysical instrumentation, which have been developed mainly to study protein folding and stability [16] but could now be used to study flexibility. While typically enzymes are studied by perturbing temperature and chemical composition, which are easy to vary in the laboratory, recently special high-pressure cells have been made for standard biophysical instruments such as NMR spectroscopy [24], X-ray crystallography [25], and X-ray [26] and neutron [27] scattering as well as various optical spectroscopies and imaging [28], calorimetry [29] and biochemical assays [19], and calorimetry as well as biochemical assays. Molecular studies are also going beyond in vitro to in vivo; for instance, incoherent neutron scattering under high pressure has been used to investigate molecular motions in intact deep sea microbes [30]. In addition, while many of the cells were limited to 3 to 4 kbar, diamond anvil cells are now available, which allow studies above 10 kbar [28]. To complement experimental studies, atomistic molecular dynamics simulations of folded proteins are particularly suited to interpret flexibility. And last but not least, the PDB has continued to provide structures of more proteins that are readily available for molecular studies.

CONCLUDING REMARKS

While much progress has been made in understanding proteins, especially their stability, a future challenge is to understand what are the essential features of their functionality. For instance, the characterization and determination of a stability-flexibility relationship as a function of P-T-X for enzyme activity would lead to a better of understanding of their activity. A novel approach would be to investigate targeted enzymes in Nature’s laboratory under various P-T-X conditions from new field studies combined with biophysical and biochemical characterization of the same enzymes at different P-T-X conditions using advanced instrumentation and computational methods. Of course, while the approach has been framed in terms of enzymes, extending it to other proteins, nucleic acids, and biomembranes is necessary for a molecular understanding of how life adapts to different conditions. Ultimately, a P-T-X diagram for life could be developed based on predictions of the conditions that biomolecules can be active rather than where organisms have been found.