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Class IIa histone deacetylases (HDAC) have been shown to drive innate immune cell-mediated inflammation in the peripheral system, but their roles in cerebral inflammatory responses remain largely unknown. Here, we elucidate that HDAC7 is selectively elevated in lipopolysaccharide (LPS)-challenged astrocytes both in vivo and in vitro. We identify that HDAC7 binds to the inhibitory kappa B kinase (IKK) to promote IKKα and IKKβ deacetylation and subsequent activation, leading to the activation of nuclear factor κB (NF-κB). Astrocyte-specific overexpression of HDAC7 results in NF-κB activation, pro-inflammatory gene upregulation and anxiety-like behaviors in mice, while downregulating HDAC7 reserves LPS-induced NF-κB activation and inflammatory responses. Furthermore, pharmacological inhibition of HDAC7 by a class IIa HDAC inhibitor attenuates LPS-induced NF-κB activation, inflammatory responses and anxiety-like behaviors both in vivo and in vitro. Together, our data reveal a novel mechanism of HDAC7 in astrocyte-mediated inflammation and suggest that targeting HDAC7 could be a potential therapeutic strategy for the treatment of anxiety and other inflammation-related diseases.

Alzheimer's disease (AD) is the most common type of dementia that occurs in the elderly. Amyloid hypothesis is one of the most studied pathological mechanisms, and β-amyloid (Aβ) is the drug target for most clinical trials. Mitochondrial dysfunction induced by the Aβ-precursor protein (APP)/Aβ has been suggested to play a key role in the development of AD. Here, we explored the effects of myricetin, a polyphenol compound abundant in fruits and vegetables, on mitochondrial damages in N2a-SW cells. After the treatment of myricetin, mitochondrial depolarization was improved by increasing the mitochondrial membrane potential. Mitochondrial biogenesis as well as mitochondrial genome integrity was enhanced via increased levels of PGC-1α, Nrf1, TFAM, and the copy number of mtDNA. Mitochondrial functions were restored as represented by the increased levels of proteins involved in the electron transport chain and the adenosine 5'-triphosphate (ATP) content and the decreased concentration of ROS. Mitochondrial dynamics and mitophagy were ameliorated through the regulation of proteins involved in fusion (OPA1 and Mfn2), fission (Drp1 and Fis1), and mitophagy (PINK1 and Parkin). Thus, it is summarized that myricetin could recover the mitochondrial impairments in N2a-SW cells, exhibiting the potential to promote neuroprotection for APP/Aβ-related diseases, including AD.

A new disease designated as Pale liver disease (PLD) has been circulating in Chinese Muscovy duck flocks since 2014, which is characterized by fatigue, diarrhoea, sudden death and acute hepatitis with pale and haemorrhagic liver. In this study, the etiological agents of PLD were isolated, causing a significant cytopathic effect (CPE) by cell rounding. Virus particles were observed by transmission electron microscopic (TEM) observation. The same disease was reproduced by experimental infection with the isolate BG61. The whole genomes of isolates were 43,842 nt in length with a GC content of 47.11%, similar to French Muscovy duck adenovirus strain GR with a GC content of 46.08%. The isolates shared 99.71-99.95% and 93.31-93.33% identity with Chinese Muscovy duck adenovirus isolates and GR strain, respectively. The DNA polymerase gene of all Muscovy duck adenovirus strains formed a separate genetic lineage with 99.55-100% amino acid sequence identity. All Chinese Muscovy duck adenovirus isolates contained two fibre genes. In contrast, only one fibre gene was found in GR, the only representative strain in species Duck aviadenovirus B. Anti-DAdV-2 serum antibodies had a weak neutralizing activity against Chinese Muscovy duck adenovirus isolates. The phylogenetic trees of the complete genome, hexon and fibre proteins revealed that all Muscovy duck adenovirus strains formed a major genetic lineage consisting of two clades. Thus, both GR and Chinese Muscovy duck adenovirus strains were proposed to be included in the same species of Duck aviadenovirus B belonging to the genus Aviadenovirus. The species Duck aviadenovirus B included two serotypes or genotypes, such as GR, which represents the strain of serotype 1 or genotype 1 (DAdV B1) and Chinese Muscovy duck adenovirus strains, which belong to serotype 2 or genotype 2 (DAdV B2).

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease threatening the health of the elderly, but the available therapeutic and preventive drugs remain suboptimal. Loganin, an iridoid glycoside extracted from Cornus officinalis, is reported to have anti-inflammatory and memory-enhancing properties. This study is aimed to explore the influence of loganin on cognitive function in 3xTg-AD mice and the underlying mechanism associated with its neuroprotection. According to the results of behavioral tests, we found that administration of loganin could significantly alleviate anxiety behavior and improve memory deficits of 3xTg-AD mice. Furthermore, immunohistochemical analysis displayed that there were decreased Aβ deposition in the hippocampus and cortex of 3xTg-AD mice treated with loganin compared with the control mice. Importantly, the Aβ-related pathological change was mainly involved in altering APP expression and processing. And loganin was also found to reduce the levels of phosphorylated tau (i.e. pTauS396 and pTauS262) in 3xTg-AD mice. By performing 2D-DIGE combined with MALDI-TOF-MS/MS, we revealed 28 differentially expressed proteins in the 3xTg-AD mice treated with loganin compared with the control mice. Notably, 10 proteins largely involved in energy metabolism, synaptic proteins, inflammatory response, and ATP binding were simultaneously detected in 3xTg-AD mice compared to WT mice. The abnormal changes of energy metabolism (PAGM1 and ENO1), synaptic proteins (SYN2 and Cplx2), inflammatory response (1433Z) were verified by western blot. Overall, our study suggested that loganin could be used as a feasible candidate drug to ameliorate molecular deficits, pathologies and cognitive impairment for prevention and treatment of AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects 50 million people worldwide. The current medicines have modest benefits in preventing or curing AD. Thus, it is urgent to discover drugs with the potential to change the progression of the disease. The primary clinical symptoms are memory loss and anxiety, while the critical pathological characteristics are Aβ plaques and hyperphosphorylated tau tangles. In this study, isobavachalcone (ISO), isolated from Psoralea corylifolia, was administered to 3×Tg-AD mice. It has been shown that this compound could significantly improve anxiety, memory and recognition deficits in the AD mice, attenuate the accumulation of Aβ oligomers, reduce the hyperphosphorylation of tau, and prevent the production of tau filaments. The metabolomic analysis implicates that the most probable pathways affected by ISO were bile secretion, tyrosine metabolism, and purine metabolism. In summary, ISO possesses the potential for further development as a drug candidate for AD.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases without any effective medicine treatments. The neurofibrillary tangles containing hyperphosphorylated tau protein are one important pathological characteristic. Thus, one practicable strategy for AD drug design is to discover compounds that could inhibit tau protein aggregation and/or phosphorylation. In this study, isobavachalcone, a natural plant-derived compound, has been shown to inhibit tau protein aggregation and disaggregate tau fibrils in vitro by directly interacting with tau protein at amino acids I278, V309, etc. It is able to reduce tau phosphorylation at four disease-related sites in vivo by regulating the critical kinase and protein phosphatase, GSK3β and PP2A. The compound also exhibits protection against tau oligomers-induced apoptosis through the mitochondria and ER mediated apoptotic pathways. In summary, isobavachalcone is a promising candidate for further evaluation as a potential preventive and therapeutic medicine for AD.

Alzheimer's disease (AD) is a serious neurodegenerative disease in people of age 65 or above. The detailed etiology and pathogenesis of AD have not been elucidated yet. In this study, the hippocampi of 2- and 6-month-old triple transgenic Alzheimer's disease male mice and age-sex-matched wild-type (WT) mice were analyzed by using targeted metabolomics approach. Compared with WT mice, 24 and 60 metabolites were found with significant differences in 2- and 6-month-old AD mice. Among these, 14 metabolites were found common while 10 metabolites showed consistent variable trends in both groups. These differential metabolites are found associated with amino acid, lipid, vitamin, nucleotide-related base, neurotransmitter and energy metabolisms, and oxidative stress. The results suggest that these differential metabolites might play a critical role in AD pathophysiology, and may serve as potential biomarkers for AD. Moreover, the results highlight the involvement of abnormal purine, pyrimidine, arginine, and proline metabolism, along with glycerophospholipid metabolism in early pathology of AD. For the first time, several differential metabolites are found to be associated with AD in this study. Targeted metabolomics can be used for rapid and accurate quantitative analysis of specific target metabolites associated with AD.

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis andin vitrophosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in β−sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.

Class IIa histone deacetylase s (HDAC) have been shown to drive innate immune cell-mediated inflammation in the peripheral system, but their roles in cerebral inflammatory responses remain largely unknown. Here, we elucidate that HDAC7 is selectively elevated in lipopolysaccharide (LPS)-challenged astrocytes both in vivo and in vitro. We identify that HDAC7 binds to the inhibitory kappa B kinase (IKK) to promote IKKα and IKKβ deacetylation and subsequent activation, leading to the activation of nuclear factor κB (NF-κB). Astrocyte-specific overexpression of HDAC7 results in NF-κB activation, pro-inflammatory gene upregulation and anxiety-like behaviors in mice, while downregulating HDAC7 reserves LPS-induced NF-κB activation and inflammatory responses. Furthermore, pharmacological inhibition of HDAC7 by a class IIa HDAC inhibitor attenuates LPS-induced NF-κB activation, neuroinflammation and anxiety-like disorders both in vivo and in vitro. Together, our data reveal a novel mechanism of HDAC7 in astroglial inflammation and suggest that targeting HDAC7 could be a potential therapeutic strategy for the treatment of anxiety and other neuroinflammation-related diseases.

Proteins fold on relatively smooth free energy landscapes which are biased toward the native state, but even simple topologies which fold rapidly can experience roughness on their free energy landscape. The details of these interactions are difficult to elucidate experimentally. Closely related to the problem of deciphering the details of the free energy landscape is the problem of defining the interactions in the denatured state ensemble (DSE) which is populated under native conditions, that is, under conditions where the native state is stable. The DSE of many proteins deviates from random coil models, but quantifying and defining the energetics of the transiently populated interactions in this ensemble is extremely challenging. Characterization of the DSE of proteins which fold to compact structures is also relevant to studies of intrinsically disordered proteins (IDPs) since interactions in the dynamic ensemble populated by IDPs can modulate their behavior. Here we show how experimental thermodynamic and pKa measurements can be combined with computational thermodynamic integration to quantify interactions in the DSE. We show that non-native side chain interactions can stabilize native backbone structure in the DSE and demonstrate that that even rapidly folding proteins can form energetically significant non-native interactions in their DSE. As an example, we characterize a non-native salt bridge that stabilizes local native backbone structure in the DSE of a widely studied fast-folding protein, the villin headpiece helical domain. The combined computational experimental approach is applicable to other protein unfolded states and provides insight that is impossible to obtain with either method alone.

Alzheimer’s disease (AD) is the most common age-associated dementia with complex pathological hallmarks. Mitochondrion, synaptosome, and myelin sheath appear to be vulnerable and play a key role in the pathogenesis of AD. To clarify the early mechanism associated with AD, followed by subcellular components separation, we performed iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics analysis to simultaneously investigate the differentially expressed proteins (DEPs) within the mitochondria, synaptosome, and myelin sheath in the cerebrum of the 6-month-old triple transgenic AD (3 × Tg-AD) and 6-month-old wild-type (WT) mice. A large number of DEPs between the AD and WT mice were identified. Most of them are related to mitochondria and synaptic dysfunction and cytoskeletal protein change. Differential expressions of Lrpprc, Nefl, and Sirpa were verified by Western blot analysis. The results suggest that decreased energy metabolism, impaired amino acid metabolism and neurotransmitter synthesis, increase compensatory fatty acid metabolism, up-regulated cytoskeletal protein expression, and oxidative stress are the early events of AD. Among these, mitochondrial damage, synaptic dysfunction, decreased energy metabolism, and abnormal amino acid metabolism are the most significant events. The results indicate that it is feasible to separate and simultaneously perform proteomics analysis on the three subcellular components.

Intrinsically disordered proteins (IDPs) possess a wide range of biological function in all organisms, however the specific functions of most IDPs are still unknown. Soybean LOC protein, LOC for short, is a heat-stable protein, which is more abundant in the stress-resistant radicles. Sequence alignment and phylogenetic analysis showed that LOC is a functionally unknown protein and conserved in Fabaceae. LOC, being enriched in most disorder-promoting residues and depleted in most order-promoting residues, was predicted to contain high levels of intrinsic disorder by several commonly used computational tools. However, it was also predicted to contain two disorder-based protein-protein binding sites and two short α-helical segments. The circular dichroism spectroscopic analysis showed that this protein is mostly disordered in water, but can form more α-helical structure in the presence of SDS and TFE. Functional in vitro studies showed that the LOC protein is able to prevent lactate dehydrogenase inactivation by freeze-thaw at a molar ratio of 10:1. Furthermore, in vivo analyses revealed the survival rate of Escherichia coli over-expressing LOC protein under the conditions of osmotic stress was noticeably increased in comparison with the control. These observations suggest that the intrinsically disordered protein LOC might serve as a chaperone and/or cell protector.

The microtubule-associated protein tau can undergo liquid–liquid phase separation (LLPS) to form membraneless condensates in neurons, yet the underlying molecular mechanisms and functions of tau LLPS and tau droplets remain to be elucidated. The human brain contains mainly 6 tau isoforms with different numbers of microtubule-binding repeats (3R, 4R) and N-terminal inserts (0N, 1N, 2N). However, little is known about the role of N-terminal inserts. Here we observed the dynamics of three tau isoforms with different N-terminal inserts in live neuronal cell line HT22. We validated tau LLPS in cytoplasm and found that 2N-tau forms liquid-like, hollow-shell droplets. Tau condensates became smaller in 1N-tau comparing with 2N-tau, while no obvious tau accumulated dots were shown in 0N-tau. The absence of N-terminal inserts significantly affected condensate colocalization of tau and p62. The results reveal insights into the tau LLPS assembly mechanism and functional effects of N-terminal inserts in tau.

Alzheimer's disease (AD) is a neurodegenerative disease. The molecular mechanisms of AD are not yet clear, and no effective treatments are available to cure AD. Because of the huge number of patients and related costs, it is urgent to discover new medicines to prevent or cure AD. In this study, xanthohumol (XN), a natural botanic compound, is found to inhibit tau protein aggregation and disaggregate tau fibrils. XN directly interacts with tau protein at sites sporadically located in all four repeating domains with a Kd value of 52 μM. Binding with XN does not alter the secondary structures of tau protein. Cellular experiments showed that XN exhibits little cytotoxicity and attenuates apoptosis induced by tau oligomers. The results provide preliminary experimental data to develop XN into medicines, food products, or nutritional supplements for AD. The findings also provide data for computational drug design.

Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single (13)C═(18)O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, (13)C═(18)O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the (13)C═(18)O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the (13)C═(18)O labels at different positions in HP36 show drastic differences in the midpoint of their transitions (T(m)), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for (13)C═(18)O A57 is 2.4e + 05 s(−1) whereas for (13)C═(18)O L69 HP36 the relaxation rate is 5.1e + 05 s(−1), two times faster. The observation of sitedependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.

The microtubule-associated protein Tau plays a central role in the pathogenesis of Alzheimer’s disease. Although Tau interaction with membranes is thought to affect some of its physiological functions and its aggregation properties, the sequence determinants and the structural and functional consequences of such interactions remain poorly understood. Here, we report that the interaction of Tau with vesicles results in the formation of highly stable protein/phospholipid complexes. These complexes are toxic to primary hippocampal cultures and are detected by MC-1, an antibody recognizing pathological Tau conformations. The core of these complexes is comprised of the PHF6* and PHF6 hexapeptide motifs, the latter in a β-strand conformation. Studies using Tau-derived peptides enabled the design of mutants that disrupt Tau interactions with phospholipids without interfering with its ability to form fibrils, thus providing powerful tools for uncoupling these processes and investigating the role of membrane interactions in regulating Tau function, aggregation and toxicity., The Alzheimer protein Tau interacts with biological membranes, but the role of these interactions in regulating Tau function in health and disease remains unexplored. Here, the authors report on the discovery and characterization of neurotoxic oligomeric protein/phospholipid complexes.

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.

The helical subdomain of the villin headpiece is the smallest naturally occurring cooperatively folded protein. Its small size, simple three-helix topology, and very rapid folding have made it an extremely popular model system for computational and theoretical studies of protein folding. The domain has a well-packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues, F47, F51, and F58 (denoted using the numbering of the larger headpiece protein). Aromatic-aromatic interactions have been thought to play a critical role in specifying the subdomain fold and have been proposed to play a general role in stabilizing small proteins. The modest stability of the subdomain has hindered studies of core packing since multiple mutations can lead to constructs which fail to fold, and even single mutants can result in poorly folded variants. Using a previously characterized hyperstable mutant of the domain, generated by targeting surface residues, a complete set of single, double, and triple core Phe to Leu mutants were characterized. A highly conserved surface Trp which is part of a Trp-Pro interaction was also examined. All mutants are well-folded as judged by CD and NMR, and all exhibit sigmoidal urea and thermally induced unfolding transitions, thus proving that aromatic-aromatic, aromatic-proline, or aromatic-hydrophobic interactions are not required for specifying the subdomain fold. Double mutant cycle analysis demonstrates that F47 and F51 make the strongest pairwise interaction. Mutations which lack F58 are the most destabilized, although even the triple mutant is folded. Interestingly, mutation of the central Phe, F51, has the smallest effect on stability even though it makes contact with both F47 and F58 and appears to form the strongest pairwise interaction.

Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer’s disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau’s poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.

Understanding the folding of the β-hairpin is a crucial step in studying how β-rich proteins fold. We have studied CLN025, an optimized ten residue synthetic peptide, which adopts a compact, well-structured β-hairpin conformation. Formation of the component β-sheet and β-turn structures of CLN025 was probed independently using a combination of equilibrium Fourier transform infrared spectroscopy and laser-induced temperature jump coupled with time-resolved infrared and fluorescence spectroscopies. We find that CLN025 is an ultrafast folder due to its small free energy barrier to folding and that it exceeds the predicted speed limit for β-hairpin formation by an order of magnitude. We also find that the folding mechanism cannot be described by a simple two-state model, but rather is a heterogeneous process involving two independent parallel processes. Formation of stabilizing cross-strand hydrophobic interactions and turn alignment occur competitively, with relaxation lifetimes of 82 ± 10 and 124 ± 10 ns, respectively, at the highest probed temperature. The ultrafast and heterogeneous folding kinetics observed for CLN025 provide evidence for folding on a nearly barrierless free energy landscape, and recalibrate the speed limit for the formation of a β-hairpin.

The helical subdomain of the villin headpiece (HP36) is one of the smallest naturally occurring proteins that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extraordinary popular model system for computational, theoretical, and experimental studies of protein folding. Aromatic-proline interactions involving Trp64 and Pro62 have been proposed to play a critical role in specifying the subdomain fold by acting as gatekeeper residues. Note that the numbering corresponds to full-length headpiece. Mutation of Pro62 has been shown to lead to a protein that does not fold, but this may arise for two different reasons: The residue may make interactions that are critical for the specificity of the fold or the mutation may simply destabilize the domain. In the first case, the protein cannot fold, while in the second, the small fraction of molecules that do fold adopt the correct structure. The modest stability of the wild type prevents a critical analysis of these interactions because even moderately destabilizing mutations lead to a very small folded state population. Using a hyperstable variant of HP36, denoted DM HP36, as our new wild type, we characterized a set of mutants designed to assess the role of the putative gatekeeper interactions. Four single mutants, DM Pro62Ala, DM Trp64Leu, DM Trp64Lys, and DM Trp64Ala, and a double mutant, DM Pro62Ala Trp64Leu, were prepared. All mutants are less stable than DM HP36, but all are well folded as judged by CD and (1)H NMR. All of the mutants display sigmoidal thermal unfolding and urea-induced unfolding curves. Double-mutant cycle analysis shows that the interactions between Pro62 and Trp64 are weak but favorable. Interactions involving Pro62 and proline-aromatic interactions are, thus, not required for specifying the subdomain fold. The implications for the design and thermodynamics of miniature proteins are discussed.