Structural Biochemistry/Protein Disorder
Protein disorder is still a fuzzy concept for scientists to solve. Nowadays, views about protein are shaped by the structures that have been solved by X-ray crystallography. However, these beautiful structures are not able to tell the dynamic properties and regions that show considerable flexibility of proteins. In fact, only 25% of crystal structures reveal more than 95% of the complete molecular structural of proteins; the others have missing electron density for regions that are usually take on multiple conformations. Additionally, the data from X-ray crystallography are biased toward those crystallizable proteins that fold into a single or a few distinct conformations.
Protein posses a wide range of stability and degree of order/disorder. The continuous spectrum of structural states span from one extreme end of globally intrinsically disordered proteins to another extreme end of well-folded and stable proteins. Due to this wide range of flexibility of the protein, it is difficult to summarize the degree of protein flexibility with a single term; terms such as intrinsically disordered and conditionally disordered are proposed to describe protein structures:
- Intrinsically disordered proteins are those proteins that lack a stable structure and show substantial disordered regions when studied as an isolated polypeptide china under physiological conditions in vitro.
- Conditionally disorder proteins are those proteins that are intrinsically disordered under some circumstance and gain order under others, such as in the presence of their biding partners. These proteins are majority of intrinsically disorder proteins.
Intrinsic disorder is commonly observed within proteins. Approximate 30% to 50% of eukaryotic proteins contain regions of more than 30 amino acid that do not have a defined secondary structure or unstructured in vitro. Although more sophisticated technology is used to determine the structure the protein, it is still challenge to verify the folding status of the protein, especially within the cell. It is still unclear that whether those proteins that have been experimentally shown to be partially or fully unfolded in vitro are really unstructured in the cell. This is because the fact that molecular crowding and the presence of the appropriate binding partners transfer many disorder proteins to their folded state. Moreover, although intrinsically disordered proteins are sensitive to proteolytic degradation in vitro, they do generally exhibit reduced half-life in vivo, possibly because they are stabilized in cell and decrease the extent of disorder. Therefore, those proteins that are defined as globally intrinsically disordered proteins from in vitro and bioinformatics-based approaches might have gain order in cell.
Various techniques distinguishing intrinsically disorder regions (IDRs) from ordered regions and provide experimental information on protein disorder. Among all different techniques, NMR is unrivaled because it is able to provide detailed residue-by-residue information on the extent of disorder, residual dipolar coupling, and paramagnetic resonance enhancement measurements. In order to determine the information on protein disorder in vivo, new technique called 'in-cell' NMR spectroscopy is developed and used to determine protein structure within living Escherichia coli cells. This 'in-cell' spectroscopy and SUPREX (stability of unpurified proteins from rates of H/D exchange) are used to establish the true in vivo extent of disorder within proteins.
Is protein disorder default?Edit
Protein disorder is often misunderstood as the default of the protein. In fact, maximum of only approximate 1 in 1010 random sequences is expected to fold into a defined structure; majority of proteins contains some regions of ordered structure, suggesting that order is selected for during evolution. Since most mutations are destabilizing, protein disorder might be simply a negative consequence of the random mutations occurred during evolution. Therefore, the frequent occurrence of disorder within proteins does not make proteins not functional. Disorder is recognized as providing functional advantages by enhancing binding plasticity, enzymatic catalysis, and allosteric coupling. Thus, disorder might in fact play an important role in molecular recognition and cellular signaling. Additionally, disorder might also increasing conformational entropy and flexibility by decreasing stability. This implies that protein disorder might play a helpful role in in vivo regulation. Lastly, studies have shown that conformational entropy conferred by disordered regions decreases the propensity of proteins to self-aggregate. Base on this fact, scientists hypothesizes that IDRs can prevent unwanted aggregation process within the crowed environment of the cell.
Conditionally disordered proteinsEdit
Conditionally disorder proteins are those proteins that can exist in at least two states, one that shows a high degree of flexibility and disorder and a second state that shows a higher degree of order. Many disordered proteins refold when they bind their partners. This is probably because the refolding is guided by thermodynamic principles that dictate that binding will stabilize and strengthen binding interactions. The order-to-disorder-to-order transitions can also occur as part of catalytic cycle of enzymes. The proteins that have only one binding site that engages multiples binding partners exemplify the concept of conditional disorder. Those 'disordered' binding sites are more likely to fold into multiple distinct conformations after binding to different partners than those 'ordered' binding sites. This fact reveals that disordered proteins have multiple distinct conformations upon binding to different partners; thus, disorder is functionally important. There are two model explaining how a partially unfolded surface regains structure by binding to different partners:
- Conformational selection hypothesis: molecular recognition mechanism based on the assumption that a small proportion of the intrinsically protein population is in appropriate configuration to interact with specific binding partner. This interaction stabilizes both the proteins and binding partners by shifting the equilibrium towards the binding competent conformation.
- Folding upon binding: molecular recognition mechanism based on the assumption that intrinsically disordered regions first bind to binding partner and then subsequently refold.
Disordered proteins may fold into different conformations by binding to different partners.A chameleon-like manner (distinct conformations) is observed when the disordered C terminus of p35 binds to various client. This observation is consistent with its functional role, which is to interact with over 40 different binding partners. However, this chameleon-like manner of intrinsically disordered proteins are rarely observed. This might be because structurally different partners for the same intrinsically disorder proteins and the structure of the protein-binding partner complexes are difficult to identify and determine.
Prototypes of proteins with multiple binding partnersEdit
Proteins that engage in multiple mutually exclusive transient interactions are more likely to have a higher degree of disorder. This high degree of disorder is also observed in chaperones, which bind to many different protein folding intermediates to prevent non-specific protein aggregation and facilitate protein folding both in vitro and in vivo. The degree of disorder of chaperones spans from 24% to 100%.
Folding chaperones, such as Hsp70, Hsp60, Hsp90, undergo large conformational rearrangements that are driven ATP-binding and ATP-hydrolysis. The intrinsic disorder enables the function of ATP-dependent chaperones by supporting dynamic conformational rearrangements necessary for client protein maturation. However, it is still unclear about the role of disordered regions in ATP-dependent chaperones.
- The highly flexible linker between nucleotide binding domain and the client-binding domain allows large interdomain conformational changes.
- The C termini that are disorder in the ATP-free apo-GroEl but are more ordered in the ATP-bound form.
- The unstructured regions provide interdomain flexibility and confer solubility to Hsp90-client complexes.Additionally, several phosphorylation sites might be involved in order-to-disorder transitions during ATP binding and hydrolysis cycle.
ATP-independent chaperones use order-to-disorder transitions to trigger activation and client binding and use disorder-to-order transitions to control client release. The stress conditions that activate these ATP-independent chaperones, including low pH and severe oxidative stress, results in unfolding of proteins, which usually leads to the inactivation. Unlike other proteins, these ATP-independent chaperones is activated by their unfolding due to the stress.
- HdeA is an acid-activated conditionally disordered chaperone that protects proteins from aggregation induced by low pH and bacteria from acidic stress. At pH 7, HdeA is well-folded dimer with no chaperone function. While shift to pH 2, HdeA is activated as a chaperone by partially unfolding and menomerizing within 2 seconds. Its nature of being partially disorder at low pH enables it be flexible of interacting with difference substrate that protects proteins from the irreversible damage; thus, it protects the bacteria from the acidic stress . The flexibility of HdeA suggests that HdeA could have the chameleon-like binding property. When the pH goes back to natural, Hdea slowly releases it client protein to minimize the aggregation-sensitive folding intermediates. Thus, the client proteins refolds back to its original structure passively while aggregation is disfavored.
- Hsp33 is an oxidative stress-activated intrinsically disordered chaperone that protects proteins from oxidative unfolding. In the inactive state, Hsp33 is a monomeric two-domain protein that contains the tetrahedral, high affinity binding between a single zinc ion and 4 absolutely conserved cysteines in far C terminus. This binding of zinc ion stabilizes the C terminus and a metastable linker region. When Hsp33 is exposed to oxidizing conditions, zinc ion is released and two disulfide bonds are formed; zinc binding domain is destabilized; the linker region is unfolded; the protein is dimerized. The unfolding of the linker region actives the chaperone function of Hsp33. After Hsp33 is activated, it uses its intrinsically disordered linker region to interact with protein folding intermediates that contain significant amount of secondary structure so that a more stabilized conformation of Hsp33 and client protein is adopted. Hsp33 protects client proteins from stress-induced aggregation and shields bacteria from the antimicrobial oxidant bleach. Upon return to non-stress, Hsp33 is refolded, and this refolding of Hsp33 triggers the unfolding of the client proteins; the affinity between Hsp33 and client proteins decreases. Then, client proteins are released to ATP-dependent chaperone foldases, in which the client proteins are bound and refolded back to their native state.
- Hsp26, which is a member of small heat shock protein, is a heat-activated conditionally disordered chaperone that protect proteins from aggregation induced by elevated temperature and bacteria from heat stress. At room temperature, Hsp26 is inactivated. Upon the induction of heat shock temperature, Hsp26 undergoes conformational changes by folding its unique thermosensing regions , and its chaperone function is activated. Unlike HdeA and Hsp33, the intrinsically disordered region of Hsp26 does not directly bind the client proteins but does interact with the client proteins.
Globally intrinsically disordered chaperonesEdit
Some chaperones that are globally disordered in vitro also have anti-aggregation activity.Globally intrinsically disorder chaperones inhibit aggregation by physically shielding and preventing folding intermediates from interacting with other aggregation-sensitive entities. Due to this fact, globally intrinsically disordered chaperones are relatively inefficient.
- Caseins shield aggregation-prone surfaces and increases refolding rates through transient hydrophobic interactions. Caseins prevent client proteins from participation by forming soluble micellar complexes with client proteins. Since caseins actively inhibit lysozyme refolding and are unable to prevent activity lose of catalase and alcohol dehydrogenase induced by heat, they are not considered as folding chaperones.
- It is unclear whether caseins play a role of chaperon in vivo or not.
- It is possible for casein to compensate its inefficiency in the presence of high concentration.
- LEA are proteins are highly hydrophilic. When LEA proteins are dehydrated under standard buffer conditions, they are activated as chaperones and adopt α-helical configurations. Similar to caseins, LEA proteins acts via transient hydrophobic interactions to shield aggregation-prone surfaces and increases refolding rates. They protect client proteins from dehydration-mediated and temperature-mediated inaction and aggregation in vitro.
- LEA proteins play a role of chaperon and folding protein in vivo.
- It is possible for LEA proteins to compensate its inefficiency in the presence of high concentration.
- α-synucleins inhibit the aggregation of protein induced by heat. The inhibition of α-synucleins is less efficient than that of small heat shock proteins.
- It is unclear whetherα-synucleins play a role of chaperon in vivo or not.
Bardwell, James C.A., and Ursula Jakob. "Conditional Disorder in Chaperone Action." Trends in Biochemical Sciences 37.23 (2012): 517-25. ScienceDirect. Web. 5 Dec. 2012. <http://www.sciencedirect.com/science/article/pii/S0968000412001272>