Biological Roles of Protein Kinetic Stability - Biochemistry (ACS

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Biological roles of protein kinetic stability Wilfredo Colon, Jennifer Church, Jayeeta Sen, Jane Thibeault, Hannah S Trasatti, and Ke Xia Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00942 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Biological Roles of Protein Kinetic Stability Wilfredo Colón1, 2, *, Jennifer Church1, 2, Jayeeta Sen1, 2, Jane Thibeault2, 3, Hannah Trasatti1, 2, and Ke Xia1, 2 1

Department of Chemistry and Chemical Biology,

Interdisciplinary Studies,

3

2

Center for Biotechnology and

Biochemistry and Biophysics Graduate Program, Rensselaer

Polytechnic Institute, Troy, New York 12180

1 ACS Paragon Plus Environment

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ABSTRACT: A protein’s stability may range from non-existent, as in the case of intrinsically disordered proteins, to very high, as indicated by a protein’s resistance to degradation, even under relatively harsh conditions. The stability of this latter group is usually under kinetic control due to a high activation energy for unfolding that virtually traps the protein in a specific conformation, thereby conferring resistance to proteolytic degradation and misfoldingaggregation. The usual outcome of kinetic stability is a longer protein half-life. Thus, the protective role of protein kinetic stability is often appreciated, but relatively little is known about the extent of biological roles related to this property. In this review, we will discuss several known or putative biological roles of protein kinetic stability, including protection from stressors to avoid aggregation or premature degradation, achieving long-term phenotypic change, and regulating cellular processes by controlling the trigger and timing of molecular motion. The picture that emerges from this analysis is that protein kinetic stability is involved in a myriad of known and yet to be discovered biological functions via its ability to resist degradation and control the timing, extent, and permanency of molecular motion.


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The stability of proteins in nature seems to be optimized to confer the most benefit to an organism. Proteins that need to be highly regulated may be marginally stable to allow quick turnover, whereas proteins that need to be long-lived are usually hyperstable to resist proteolytic degradation. Most soluble proteins in biological systems appear to be marginally stable, which likely allows their efficient removal or regulation as needed. However, protein stability may range from being non-existent, as in the case of intrinsically disordered proteins (IDPs), to being extremely high. Whereas the biological roles of IDPs have been increasingly studied over the past 15 years,1 the extent of biological roles of protein hyperstability or degradation-resistance is not as well understood. The stability of soluble proteins is known to be under either thermodynamic control (TC) or kinetic control (KC), with the former being predominant. Folded proteins whose stability is under TC are in equilibrium with their partially and globally unfolded states, and their stability is quantitatively defined as the difference in Gibbs free energy between the native and denatured states (∆GU) (Figure 1A). The stability of proteins under KC is not defined by ∆GU, but rather by their activation energy for unfolding (∆GU≠), which virtually traps them in a specific structure (Figure 1B). The high unfolding energy barrier of proteins under KC causes them to unfold very slowly. Therefore, these hyperstable proteins are known as kinetically stable proteins (KSPs). Proteins with high kinetic stability (HKS) rarely explore partially and globally unfolded states within the biologically relevant time frame, and are consequently resistant to proteolysis, misfolding-aggregation, and harsh detergents2. Thus, the conformational resilience of KSPs is known to have a protective role leading to a much longer protein half-life, especially under harsh conditions.3 Interestingly, recent studies have shown that membrane proteins may also possess


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Figure 1. Free energy diagrams illustrating the different types of protein stability. (A) The thermodynamic stability of proteins is defined as the difference in free energy (∆GU) between the native (N) and unfolded (U) states. The low transition state free energy for unfolding causes the N and U states to exist in equilibrium, which favors the N state. (B) Kinetically stable proteins (KSPs) are characterized by having a high activation energy (∆GU≠) for unfolding that practically confines them in their native state (N). The stability of such proteins is under kinetic control, as their unfolding rate is very slow, which results in a longer half-life.

HKS, suggesting that HKS may play a role in protecting membrane proteins against irreversible denaturation and perhaps in regulating its biological function.4, 5 While the thermodynamic stability for most soluble proteins may be easily determined and allow for comparison among proteins, such quantitative analysis is less straightforward when analyzing kinetic stability (KS). For example, relatively few KSPs have been studied to obtain their activation energy for unfolding, which may be the most appropriate quantitative approach for determining and comparing KS among proteins. However, a valid comparison of unfolding activation energy among proteins should be performed at the same working temperature;


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otherwise, the dependence of the activation free energy with temperature must be considered. Therefore, most KSPs are identified by properties such as slow unfolding, slower rate of activity loss at high temperature, proteolytic resistance,6 slow equilibration during unfolding experiments, hysteresis during refolding experiments, and resistance to the detergent sodium dodecyl sulfate (SDS).7 This diversity of methods has resulted in a growing number of putative KSPs, even when in many studies they have not been quantitatively classified as such. What type of biological roles or functions may require proteins to possess high kinetic stability? Protein kinetic stability likely confers selective advantage to organisms and plays important biological roles beyond protection from harsh conditions. Kinetic stability may allow regulation of protein function by trapping thermodynamically unstable or metastable conformations until a particular function is needed. For example, HKS may control the timing of a protein’s transition from an inactive to active conformation via ligand binding, posttranslational modifications (PTMs), or chemical changes in the protein environment. A variety of biological roles for protein KS have been slowly emerging over the past 20 years and more are likely to arise. This review will focus on some examples illustrating the breadth of biological roles involving protein HKS. KINETIC STABILITY PROTECTS PROTEINS AGAINST HARSH CONDITIONS AND PREMATURE DEGRADATION Proteases are expressed under diverse circumstances, and most proteins are susceptible to proteolytic degradation. Proteases themselves are often susceptible to autoproteolysis and cleavage by other proteases, the former often serving as a mechanism of autoregulating protease activity. However, some proteins, including many proteases, must be resistant to proteolysis in order to have a longer half-life, which may be achieved by possessing HKS (Figure 2). The


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folded state of proteins act as poor substrates for effective proteolysis, which requires that the target cleavage site be accessible.6 For non-KSPs, efficient proteolysis is possible because even under native conditions the unfolding rate (ku) of proteins is fast enough to allow transient unfolding. On the contrary, the high-energy barrier of KSPs decreases their unfolding rate and prevents frequent sampling of cleavable states. One of the best-studied kinetically stable proteases is the extracellular bacterial protein alphalytic protease (α-LP).8, 9 Interestingly, unlike many other proteases that fold spontaneously to a

Figure 2. High kinetic stability increases the proteolytic resistance of proteins. (A) Proteins whose stability is under thermodynamic control frequently sample partially and globally unfolded states, giving rise to their susceptibility to proteolysis. (B) In contrast, the high unfolding energy barrier of KSPs causes them to rarely expose the extended backbone chain that is necessary for proteolysis.


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thermodynamically stable state, the native state of α-LP is thermodynamically unstable.9 α-LP is synthesized as a proenzyme form containing a pro-region, which assists in the folding process. This region is subsequently autocleaved, leaving α-LP kinetically trapped in its native state.8 A study comparing the degradation susceptibility of bacterial α-LP and its thermodynamically stable mammalian homologues, chymotrypsin and trypsin, showed that the latter were degraded at rates 100-fold faster than α-LP.9 The environment that α-LP is exposed to as an extracellular protease in Lysobacter enzymogenes is likely harsher than that encountered by trypsin and chymotrypsin, thereby accounting for α-LP’s biological need for higher KS. In general, this type of protection may be more critical for prokaryotic than eukaryotic organisms, as suggested by a proteomic assessment of KSPs showing that prokaryotic organisms, in particular thermophiles, have more KSPs than eukaryotic organisms.10 Additionally, among the proteins of an organism, periplasmic and secreted proteins are particularly at risk from exogenous stress, and are perhaps more likely to be kinetically stable. Proteolytic resistance may be necessary for various biological reasons. For example, some proteins in beans play a role in biodefense and must resist biotic stress such as digestion by pests.11 Interestingly, arcelin and alpha-amylase inhibitor are both abundantly found in lima bean and have been shown to be kinetically stable.12 Arcelin is responsible for lima bean’s resistance against the Mexican bean weevil by binding to the chitin of the insect’s digestive system.11 If arcelin were not kinetically stable, the insect’s digestive system would degrade the protein and it would afford no protection to lima bean. Similarly, alpha-amylase inhibitor also acts on the digestive system of pests to prevent the metabolism of carbohydrates, and makes cowpea and adzuki bean resistant to weevils.13, 14 Thus, KS is the main property of proteins that determine their proteolytic susceptibility.


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Proteins that protect against oxidative stress may also require HKS. Oxidative damage induced by reactive oxygen species (ROS) is among the most ubiquitous stress encountered by all organisms in nature. Oxidative damage involves extremely fast chemical reactions and is a constant danger. Therefore, all organisms have developed defenses against ROS damage by evolving long-lived oxidative stress-response proteins, such as superoxide dismutase (SOD) and catalase (CAT). The HKS of such proteins could confer two advantages to the organism. First, these proteins would be long-lived, making it biologically economical to synthesize them in abundance and have them on “stand-by” for immediate protection against oxidative stress. Second, HKS would protect these proteins against premature inactivation caused by ROS. In the absence of HKS, proteins that are damaged by ROS may not be able to refold after transiently sampling unfolded conformations, leading to their inactivation and degradation. In contrast, kinetically stable free-radical scavenger proteins could maintain their function for a longer period even if oxidatively damaged. Superoxide dismutase is an essential antioxidant defense protein that exists in most living cells exposed to oxygen. SOD catalyzes the dismutation of the superoxide (O2−) radical into either O2 or hydrogen peroxide (H2O2), which then is degraded by other enzymes, such as CAT. The superoxide radical is produced as a by-product of oxygen metabolism and, if not regulated, causes many types of cellular damage. SOD is a highly conserved protein, and it has been shown to be kinetically stable in organisms ranging from bacteria to humans.2,

15-18

SOD from

Salmonella enterica barely loses activity after incubation with Proteinase K for 1 hr15 and SOD from Sulfolobus acidocaldarius is resistant to protease V8.16 Like SOD, CAT is a common enzyme found in nearly all organisms exposed to oxygen and protects against ROS. CAT from Aspergillus nidulans is resistant to 2% SDS, 9M urea, and reducing agents,19 whereas CAT from


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Rhodobacter sphaeroides is resistant to 10% SDS and 7M urea.20 These experimental results strongly suggest that the HKS of these highly conserved defense proteins is a property that is vital to their protective role. KINETIC STABILITY MAY BLOCK PROTEIN AGGREGATION The risk of premature protein inactivation may not just come from external stress. It appears most proteins have the capability to aggregate due to the ubiquitous presence of hydrophobic residues and the ability of the peptide backbone to form inter-chain hydrogen bonds.21 Organisms have adapted to this hazard by selecting against proteins that are highly aggregationprone under natural conditions22 and by evolving protein quality control systems.23

24

Yet many

proteins are still prone to aggregation and all organisms must deal with this constant threat. Protein HKS may have arisen as an additional mechanism for maintaining protein functionality and protecting the host by preventing the misfolding and aggregation of certain proteins (Figure 3). This seems to be evident by the association between the mutation-induced loss of KS and the aggregation of certain proteins linked to human diseases. A classic example is the ensemble of amyloid diseases associated with the tetrameric KSP transthyretin (TTR). Many missense mutations destabilize TTR and lead to the diseases familial amyloid neuropathy and familial amyloid cardiomyopathy. For TTR, the kinetic barrier is not an unfolding barrier, but rather a dissociation barrier of the native tetramer.25 Many TTR mutations cause the tetramer dissociation energy barrier to decrease allowing population of a monomeric aggregation-prone species that self-assembles into amyloid fibrils.25 Amyotrophic lateral sclerosis (ALS) is a well-known fatal disease characterized by progressive deterioration of the motor neurons. Mutation in the protein superoxide dismutase 1 (SOD1) is associated with a familial form of ALS (FALS). SOD1 is a homodimeric enzyme


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Figure 3. High kinetic stability may protect proteins from aggregation. (A) Proteins that are susceptible to misfolding and aggregation are often highly kinetically stable to limit the occurrence of aggregation-prone conformations. (B) Mutations to these KSPs could compromise their stability, thereby causing an increased population of misfolded species, which may itself be pathogenic or aggregate into a pathogenic form.

containing one copper ion and one zinc ion per subunit. SOD1 has been shown to be active under a range of denaturing conditions,26, 27 and is both thermodynamically28 and kinetically stable in the wild type holo form.17 Numerous SOD1 mutations have been found to cause FALS via a gain-of-function effect linked to its misfolding and aggregation.29 Yet the exact molecular mechanism by which SOD1 mutations lead to FALS remains unclear.27 Nevertheless, the ubiquitous nature of SOD1 aggregates in FALS, the HKS of holo-SOD1, and the aggregationprone nature of metal-deficient and many other mutants of SOD1 suggest that these features are related. It has been shown that metal-deficient wild type SOD1 and FALS-related mutants thereof are highly prone to aggregation, whereas many SOD1 mutants may retain wild type-like 10 ACS Paragon Plus Environment

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KS if properly metallated.17 Thus, it appears the loss of KS of wild type or mutant SOD1 throughout its lifetime in motor neurons will increase the risk of aggregation or abnormal interactions. Although SOD1 is present in all cells, the relatively long journey of SOD1 through neuronal axons may explain why a diminished KS of SOD1 represents a unique risk for motor neurons. In this context, it should be emphasized that the correct metallation of SOD1 is critical for its HKS, and therefore, any occurrence that compromises SOD1 metallation, even in wild type SOD1, will likely represent a risk of ALS.17 This is consistent with the idea that wild type SOD1 may be involved in sporadic cases of ALS. 29, 30 While only two examples are discussed above, protein misfolding-aggregation is associated with over 40 human diseases,31 and is a problem encountered by all living organisms. Interestingly, it has been shown that protein aggregation is a main contributor of bacteria aging.32 Thus, although it is not clear how many proteins may possess HKS for the main purpose of protection from misfolding-aggregation, it is likely many such cases exist throughout living organisms in all kingdoms. KINETIC STABILITY AND PRION PERSISTENCE Prion proteins are characterized by their ability to undergo a monomer to oligomer transition, with the latter possessing the ability to self-replicate by catalyzing the conformational change. This transition is irreversible due to the remarkable HKS of the oligomer, which is able to assemble into highly stable fibrils that are resistant to detergents and proteases (Figure 4).33-36 Prions were originally discovered as the causative agent in scrapie, a form of transmissible spongiform encephalopathies, but were subsequently found in yeast functioning as a mechanism of phenotypic inheritance and cell survival in yeast.37,

38

The most studied yeast prion is the

protein Sup35, which functions as a translation-termination factor. During starvation Sup35


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Figure 4. The role of high kinetic stability in prion formation and persistence. A cellular priongenic protein is represented by a predominantly alpha-helical structure, while the prion conformation has a beta sheet fold. Prion formation is kinetically blocked by a large energy barrier. Once the prion conformation is formed, it is highly kinetically stable and resistant to degradation. The persistent prion is then able to catalyze the conformational change of the precursor protein, thereby resulting in a highly kinetically stable and self-replicating aggregate.

switches from the non-prion form [psi-] to the prion form [PSI+], which is highly stable, SDSresistant, and proteolysis-resistant – all hallmark signs of HKS.39

40

The sequestration of

functional Sup35 proteins into kinetically stable aggregates results in a persistent loss-offunction and increase in stop codon read-through.40 A remarkable aspect of the Sup35 switch from [psi-] to [PSI+] in yeast is that it can be inherited by daughter cells during budding, and therefore the phenotype can be inherited as well. Thus, HKS contributes as a vector for epigenetic inheritance since without transmission of kinetically


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stable aggregates the related phenotype cannot be inherited by daughter cells. In the daughter cells, the prion [PSI+] will continue forming persistent fibrils and allowing translation past stop codons, thereby acting as a protein-based system of phenotypic inheritance. Intriguingly, prion-like proteins have also been implicated in the persistence of long-term memory. In Drosophila, a specific cytoplasmic polyadenylation element-binding protein (CPEB), Orb2, was found to influence long-term memory.41, 42 When a synapse is activated its intracellular composition is altered, including the protein content. CPEBs are known to regulate activity-dependent protein synthesis in the synapse, and are linked as regulators of long-term memory. Orb2 from Drosphila possesses a prion-like domain near the N-terminus and the ability to self-replicate and aggregate. Functionally, Orb2 regulates protein turnover at the activated synapse. Unlike Sup35, which is inactive upon oligomerization, Orb2 is functionally important in its oligomeric state. When neurons are stimulated, the abundance of Orb2 oligomers increases. The oligomers bind to mRNA and activate translation of proteins involved in long-term memory, which includes proteins necessary for synaptic growth and plasticity. When the oligomerization is disrupted, there is a loss of long-term memory. For example, a point mutation in Orb2, which inhibits its aggregation, was found to reduce memory persistence in Drosophila.42 The potential role of KSPs in long-term memory is intriguing. In the case of Orb2, the monomer is unstable and lacks HKS. However, upon post-translational modification, Orb2 gains stability and the capability to oligomerize.43 The oligomeric state of Orb2 is resistant to heat and SDS, indicating HKS.41 Prion proteins represent an epigenetic mechanism for regulating phenotype. These proteins may be present in different organisms to regulate diverse biological functions requiring protein conformational persistence to achieve long-term phenotypic changes. In the case of Sup35, the


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loss-of-function created by kinetically stable aggregates directly influences adaptability and survivability in yeast exposed to stress. In contrast, Orb2 becomes biologically active and, most importantly, retains activity as a kinetically stable oligomer. These are only two examples of the role of kinetically stable aggregates, but with the recent focus on discovering non-pathogenic prion proteins there will likely be many more. For example, recently a prion-like protein that functions as part of the vernalization pathway was discovered in the plant Arabidopsis.44 Thus, it is likely that kinetically stable prions are more ubiquitous in nature than previously thought. KINETIC STABILITY FOR “SPRING-LOADED” CONFORMATIONAL CHANGE “Spring-loaded” is a term that describes proteins capable of undergoing a quick spring-like conformational change. These proteins typically fold to a metastable “spring-loaded” conformation that is trapped by a kinetic barrier. Upon interaction with a trigger, the energy barrier is lowered, allowing the native protein to quickly and irreversibly transition to a lower energy state conformation. The HKS of a spring-loaded conformation is critical to maintain the protein in a tense state, primed for major structural change, which may be triggered in vivo and in vitro by factors such as heat, ligands, and changes in pH.45-47 The most well studied example of a spring-loaded protein is hemagglutinin (HA). HA is a viral surface coiled-coil trimeric glycoprotein responsible for the uptake of influenza virus by a host cell.45 HA is able to maintain a native metastable fold until it reaches the endosome of the host. The acidic environment (i.e. pH 5) within the endosome triggers a spring-loaded conformational change in HA that facilitates the fusion of the viral membrane to the endosome membrane, thereby allowing the virus genetic material to enter the host cytosol.45, 48 It has been previously demonstrated that HA is kinetically trapped in a spring-loaded metastable state, as the structural change to the fusogenic conformation may also be triggered by urea and heat at neutral pH.45


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Figure 5. The effect of high kinetic stability on spring-loaded protein conformational change. (A) A high kinetic barrier traps the protein in a metastable spring-loaded nonfunctional (NF) state. The protein exclusively exists in the NF state leading to an inability to perform a particular function (e.g. fusogenic activity). (B) The presence of a trigger (e.g. pH in the case of HA) lowers the kinetic barrier causing a quick conformation change to take place. This allows the protein to “spring” into a new functional (F) conformation.

Furthermore, native HA exhibits proteolytic resistance and SDS resistance indicating it possesses elevated KS (Figure 5).46, 49 There are several other prominent examples of kinetically trapped spring-loaded proteins. SNAREs (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors) make up a family of proteins responsible for membrane fusion that exhibit SDS-resistance when forming complexes with other proteins.50,

51

These protein complexes require disassembly for

SNARE recycling, which is accomplished through binding to the spring-loaded protein NSF. Upon binding to SNARE, NSF undergoes a spring-loaded conformational change that generates


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enough mechanical force to break apart the SNARE complex.47 Another example of a springloaded protein is cellular inhibitor of apoptosis protein-1 (cIAP1), a ubiquitin ligase that acts as a binary switch to inhibit caspase activity, thus preventing cell death. cIAP1 is natively found in a metastable monomeric closed state. A spring-loaded conformational change triggered by binding to a peptide allows cIAP1 to quickly open. With its hydrophobic domain now exposed, it dimerizes to act as a controlled regulator of apoptosis.52,

53

Other proteins, including the

transcription factor NusG, natriuretic peptide receptor, phosphoglycerate kinase, hepatitis C virus NS3 helicase, and the periplasmic chaperone Skp regulate function via a spring-loaded mechanism.54-59 The number of known spring-loaded conformations is continually increasing and has shown to manifest in a variety of organisms from viruses to yeast to mammals.45, 51, 54 In each case, the spring-loaded protein is kinetically trapped in a metastable state until a large-scale conformational change is triggered. The trigger mechanism is controlled so that the kinetic barrier is only overcome, and a fast structural change only occurs at the appropriate moment and location. The observation that diverse biological functions, from viral fusion to cell apoptosis, may be regulated by spring-loaded proteins, suggests that this strategy of controlling protein dynamics may be widespread to regulate the timing and on-off switching of protein function. KINETIC STABILITY AND THE CIRCADIAN CLOCK A large number of biochemical, physiological and behavioral changes in organisms maintain an association with time. Many periodic rhythms roughly match the day-night cycle and remain synchronized even in the absence of external stimuli.60 This phenomenon, termed circadian rhythm, is controlled by endogenous oscillators or the circadian clock, and involves the interplay of many proteins that modulate their interactions, stability, and turnover. Some of these proteins


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may be structurally trapped, thereby requiring a periodic trigger-based conformational change to switch function on or off in harmony with the clock-period. Since HKS could temporally trap a protein in a specific conformation,2 the stability of circadian proteins in some organisms may be kinetically controlled. Kinetic stability appears to play a key role in the interaction between the circadian clock and UV signaling pathways in the model plant Arabidopsis thaliana. UV Resistance Locus 8 (UVR8) has been recognized as the plant photoreceptor responsible for perceiving light signals in the UV-B region using its intrinsic tryptophan residues as chromophores.61 In order to maintain the correct synchronization between the timing generated by the various clock components and the changing day-night photoperiod in nature, clock resetting or entrainment is crucial and is brought about by the light dependent UVR8 protein. UVR8 acts as the sensor for UV-B radiation, which at low intensity, triggers a number of signaling pathways.62 UVR8 exists as a highly stable and inactive homodimer that is stabilized by a salt-bridge network.63, 64 When UVR8 is exposed to UV-B radiation the stabilizing electrostatic interactions are disrupted and the protein dissociates to an active monomer that further interacts with other proteins to regulate gene expression changes. The UVR8 dimer is SDS-resistant, indicating that HKS is trapping the dimer in the inactive state, requiring a trigger (i.e. UV-B exposure) to allow the formation of the active monomer.63, 64 Maintaining the” trapped” dimeric state in the absence of UV-B radiation (i.e in the dark) is essential to restore UV-B responsiveness and stop UV-B signaling (Figure 6). The HKS of UVR8 shuts off signaling by keeping the active monomer from being transiently populated, as would occur if the stability of the UVR8 dimer were under only thermodynamic control (Figure 1).65 It should be noted that the UVR8 dimer does not seem to exist in a spring-loaded metastable conformation, since in the absence of light, the monomer


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quickly reverts to the dimeric structure.64,

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Therefore, it appears the UVR8 dimer is

thermodynamically and kinetically stable in the dark. Upon absorption of UV-B radiation, disruption of the interactions that stabilizes and traps the dimer decreases the dissociation barrier, leading to the more stable monomer during daylight. If this proposed mechanism of dimer-

Figure 6. The putative role of high kinetic stability in maintaining the function of the UVR8 protein in the circadian clock of Arabidopsis. The highly kinetically stable and SDS-resistant UVR8 dimer is converted to the monomeric form in presence of sunlight/UV radiation, which acts as a trigger to destabilize the dimer and thereby decrease the high activation barrier blocking the dimer to monomer conversion. The active monomer then triggers a large number of signaling pathways. The monomer is converted back to the more stable dimeric state in the absence of sunlight to restore UV responsiveness. monomer switching is correct, then the HKS of the dimer is largely determined by the low free energy of the dimeric state (Figure 6). It seems plausible that HKS may play an important role in the circadian clock of other organisms. In the cyanobacteria circadian clock, proteins KaiA, KaiB and KaiC proteins are the 18 ACS Paragon Plus Environment

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three main constituents that generate a circadian rhythm of KaiC phosphorylation.67 KaiA promotes kinase activity during the day leading to autophosphorylation of KaiC. KaiB functions as a metamorphic protein, flipping between two distinct 3D folds to provide the time delay that is required to match the timing of the oscillator.68 At dusk, KaiB switches from an inactive tetramer to an active monomer and interacts with phosphorylated KaiC. The KaiB-KaiC complex binds to and inhibits KaiA, which appears to undergo a hemagglutinin-like spring-loaded conformational change, leading to KaiC autodephosphorylation.69, 70 Hence, it is likely that the HKS of the active dimeric KaiA and inactive tetrameric KaiB structures populated during the day is responsible for blocking the transient population of the structures involved in the KaiA-KaiB-KaiC night complexes. CONCLUSION AND FUTURE PROSPECTS The marginal stability of most proteins is essential for the regulation of cellular function and the degradation of proteins. Organisms must be able to quickly degrade proteins to regulate their function or to remove damaged proteins. However, there is growing evidence that the stability of certain proteins must be kinetically controlled for different biological reasons. HKS has been mostly appreciated as a means to protect proteins against external factors, including proteolysis and harsh environmental conditions. HKS is also known to protect certain proteins from aggregating by blocking their intrinsic ability to access aggregation-prone conformations. Yet, the examples reviewed here show that protein HKS may also play important roles in biological function by controlling the timing, extent, and permanency of conformational changes. HKS can trap inactive metastable states until a trigger (e.g. ligand, post-translational modification or other condition) induces a rapid and often large-scale structural change to regulate a wide-range of functions – from viral fusion to regulation of apoptosis; and the list is growing. HKS is also


Biochemistry

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integral to prion function, an epigenetic mechanism of establishing phenotypes ranging from yeast adaptation to the establishment of memory. A novel role for protein KS in circadian rhythm is proposed here for the UV signaling pathways in Arabidopsis thaliana. Since HKS is ideally suited to control the timing of conformational changes, a general role for HKS in circadian clock seems likely. Such kinetic control of conformational changes may involve a fast-acting springloaded mechanism or a binary switch involving a change in the relative stability of two protein conformations and the energy barrier between them. Overall, the existence and manipulation of high-energy barriers separating different protein conformations has evolved as a powerful method for protecting an organism and for regulating protein function. Examples of the latter continue to grow and other types of biological functions controlled by protein HKS are likely to be discovered. It also seems probable that depending on the life-time of an organism or the timescale of biological processes, even moderate protein KS in vivo – in contrast to the HKS described here - may play important functional roles yet to be discovered. Ultimately, elucidating the rules of life is going to require a comprehensive understanding of how the range of protein structures – from intrinsically disordered to highly rigid – and the range of protein stabilities – from no stability to high kinetic stability – contribute to enable, protect, and sustain life.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Wilfredo Colón: 0000-0001-6599-0218 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Financial support for this work was provided by a grant (1158375 to W.C) from the National Science Foundation, an NSF Graduate Research Fellowship to H.S.T., and a NIH Training Grant Fellowship (5T32GM067545-13) to J.T. Notes The authors declare no competing financial interest.


Biochemistry

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For Table of Contents Use Only Title: Biological Roles of Protein Kinetic Stability Wilfredo Colón, Jennifer Church, Jayeeta Sen, Jane Thibeault, Hannah Trasatti, and Ke Xia


Biological Roles of Protein Kinetic Stability - Biochemistry (ACS

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