Mechanical Stability Of Proteins Research Articles

Thermal stability of cytochrome c' from mesophilic Shewanella amazonensis.

Cytochrome c' (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c' (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c' were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c' including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures.

Covalent Incorporation of Trehalose within Hydrogels for Enhanced Long-Term Functional Stability and Controlled Release of Biomacromolecules.

Hydrogels with covalently incorporated trehalose are synthesized using thiol-ene Michael addition. Trehalose hydrogels afford prolonged stabilization and -controlled release of model enzymes in vitro and in vivo as well as preservation of protein stability under heat and -lyophilization stressors. Strong and -ordered hydrogen bonding interactions within covalently incorporated trehalose hydrogels represent a possible mechanism for protein stabilization.

Protein stabilization utilizing a redefined codon.

Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

NMR Analysis of Tuning Cross-Strand Phe/Tyr/Trp-Trp Interactions in Designed β-Hairpin Peptides: Terminal Switch from L to D Amino Acid as a Strategy for β-Hairpin Capping.

Interaction among the side chains of aromatic amino acids is a well-known mechanism of protein and peptide structure stabilization, particularly in β sheets. Using short β-hairpin models bearing the sequence Ac-Leu-Xxx-Val-DPro-Gly-Leu-Trp-Val-NH2, we report the surprising observation of significant destabilization in aryl–tryptophan interactions, which results in poorly folded peptide populations accompanied by lowering of stability. We find that such destabilization arises from forced occupancy of the indole ring in the shielded Edge position, in T-shaped aryl geometries. We demonstrate that this destabilizing effect can be efficiently salvaged by replacing the N-terminal LLeu with DLeu, which causes an increase in the folded hairpin population, while retaining Trp in the Edge position. Our observation of unique cross strand NOEs and data from temperature-dependent NMR and CD measurements reveals the formation of a locally stabilized aliphatic–aromatic network, leading to an overall increase in ΔGF° by ∼ −0.6 to −1.2 kcal/mol. Our results suggest that a contextual evaluation of stabilization by tryptophan is necessary in β hairpins. Furthermore, we report for the first time that the use of D isomers of aliphatic amino acids at the terminus is stabilizing, which can serve as a new strategy for increasing β-hairpin stability.

Mechanical stability analysis of the protein L immunoglobulin-binding domain by full alanine screening using molecular dynamics simulations.

This article is the first to study the mechanical properties of the immunoglobulin-binding domain of protein L (referred to as protein L) and its mutants at the atomic level. In the structure of protein L, each amino acid residue (except for alanines and glycines) was replaced sequentially by alanine. Thus, 49 mutants of protein L were obtained. The proteins were stretched at their termini at constant velocity using molecular dynamics simulations in water, i.e. by forced unfolding. 19 out of 49 mutations resulted in a large decrease of mechanical protein stability. These amino acids were affecting either the secondary structure (11 mutations) or loop structures (8 mutations) of protein L. Analysis of mechanical unfolding of the generated protein that has the same topology as protein L but consists of only alanines and glycines allows us to suggest that the mechanical stability of proteins, and specifically protein L, is determined by interactions between certain amino acid residues, although the unfolding pathway depends on the protein topology. This insight can now be used to modulate the mechanical properties of proteins and their unfolding pathways in the desired direction for using them in various biochips, biosensors and biomaterials for medicine, industry, and household purposes.

How Can a Free Amino Acid Stabilize a Protein? Insights from Molecular Dynamics Simulation

Amino acids such as proline are known as natural osmolytes that can stabilize proteins. But, there is no distinct mechanism at the molecular levels that can explain the stabilization effects of such osmolytes. In this paper, for the first time, the molecular level mechanism of protein stabilization by amino acid proline was studied using molecular dynamics simulation, which led to the following interesting points. Simulations were performed at room and high temperatures using lysozyme as a protein model. Although lysozyme was partially unfolded in the aqueous solution at high temperatures, the structure remained relatively native in the presence of 3 mol·L−1 proline. Protein residues showed slower dynamics in aqueous proline solution than that in water. These results are in agreement with experimental observations that show enhanced protein thermal stability in the presence of proline. It was also found that proline molecules are excluded from the protein surface and water molecules in the hydration shell around the protein increase, thus inducing thermodynamic stabilization of the protein. Moreover, it was shown that proline molecules form molecular aggregates in the solution, in agreement with previous experimental observations. We found that these aggregates can increase the order of water molecules and also the water–water hydrogen bond strength. This also leads to higher thermal stability of the protein in such solutions. The results of this paper shed some light on the molecular mechanism of protein stabilization by natural amino acids.

Mechanical Aspects of Protein Thermostability

Because thermophilic proteins are stable and functional at high temperature but generally lack activity under ambient conditions, it was suggested that thermal stability can correspond to enhanced mechanical rigidity. While this concept has initially found wide experimentally support, its generality has been later questioned. It is then natural to inquire the correlation between protein thermal and mechanical stabilities using single-molecule force-spectroscopy experiments. Here, we will present results of a combination of computational techniques, mainly based on Molecular Dynamics simulations, to tackle these problems for several mesophilic/thermophilic homologues of different molecular complexity.By applying innovative network analysis to map the protein conformational landscape, we first question the universality of the rigidity paradigm. In fact we show that for several paris of homologues, thermophiles can be as flexible as their mesophilic variant with important effects coming from the local partition of rigid/flexible regions, protein oligomerization state, or the time and length scales considered.We will then focus on the correlation among thermostability and mechanical resistance. The object under investigation is a pair of mesophilic/thermophilic cold-shock proteins for which experimental data are available. We have first implemented an enhanced-sampling algorithm which allows us to explore the proteins’ free-energy landscape over a wide range of temperatures, and we have used constant-force steered MD to investigate the effect of mechanical force. Our results suggest that thermal stability does not guarantee a mechanical one and that the associated unfolding mechanisms, of which our simulations provide extensive details, are very different. We also aim at identifying the weak-points of the protein mechanical stability and how more stable mutants can be designed. If such mutants also exhibit increased thermal stability, our approach could prove useful in numerous applications.

Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80

Detection of enhanced surface tension depression by surfactant in the presence of protein was recently suggested as a basis for determining whether protein stabilization by that surfactant is owing to surfactant forming a steric barrier at interfaces or surfactant association with the protein. In particular, protein interaction with surfactant aggregates may lead to an increased concentration of monomers thus enhancing surfactant adsorption, or to formation of surfactant–protein complexes having little or no effect on adsorption. We compared the initial rates of surface tension depression by poloxamer 188 and polysorbate 80 (PS 80) in the presence and absence of a human recombinant factor VIII (rFVIII). Indirect evidence had suggested poloxamer 188 enters into stable associations with rFVIII in solution but does not form a steric barrier at the interface, while PS 80 behaves in contrary fashion. In this study, we show the presence of rFVIII caused an increase in the rate (reduction in the activation energy) of PS 80 adsorption, while no such change was recorded in the case of poloxamer 188. Thus, we provide substantiation for detection of protein-mediated acceleration of surfactant adsorption as a means to compare different surfactants in relation to their favored mechanism for protein stabilization.

Mechanistic insights into osmolyte action in protein stabilization under harsh conditions: N-methylacetamide in glycine betaine–urea mixture

Glycine betaine (GB), a small naturally occurring osmolyte, stabilizes proteins and counteracts harsh denaturing conditions such as extremes of temperature, cellular dehydration, and presence of high concentration of urea. In spite of several studies on understanding mechanism of protein stabilization and counteraction of these harsh conditions by osmolytes, studies centred on GB, one of the most important osmolyte, are scarce, hence, there is need for more investigations. To explore mechanism of protein stabilization and counteraction of denaturing property of urea by GB, molecular dynamics studies of N-methylacetamide (NMA), a model peptide representing denatured state of a protein, in the presence of GB, urea, and GB–urea mixture were carried out. The results show that GB and urea work such that the strength of GB as a protecting osmolyte is increased and the denaturing ability of urea is decreased in the GB–urea mixture. It can be inferred that GB counteracts urea by decreasing its hydrophobic interactions with proteins. The mutual interactions between GB and urea also play an important role in protein stabilization. This study provides insights on osmolyte induced counteraction of denaturing property of urea.

Evaluation of etanercept stability as exposed to various sugars with biophysical assessment

Even though sugars have been used widely as additives for protein formulations, their exact mechanisms of protein stabilization and applicability remain still in need of investigation. The main purpose of this study was to evaluate the effects of various sugars on the biophysical stability of etanercept (Enbrel®). Six well known sugars including glucose, fructose, maltose, sucrose, trehalose, and raffinose were incorporated into the protein solution with different concentrations. The samples were analyzed with dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and size-exclusion chromatography (SEC). The DLS measurement showed that as the number of simple sugars and solution concentration increased, the hydrodynamic size increased with a decreasing absolute zeta potential. The DSC result provided consistent trends with the DLS data. As the concentration of sugar increased, the protein transition temperature (Tm) was gradually increased in most of samples. In addition, a non-enzymatic browning reaction (NEB) was observed during heating of the sugar solution. To monitor the storage stability, sample solutions were stored at 4 and 40°C. At 4°C, the ratio of monomer, aggregate, and fragment were not significantly changed. However, fragmentation of etanercept was observed in accelerated storage. In addition, fructose and maltose showed a peak shift in the SEC result. Those results suggest that the reducing ability of sugar might be a reason for the different etanercept degradation pathways. Therefore, sugars need to be carefully considered to achieve the maximum efficiency of therapeutic proteins for the development of protein formulations.

Ca2+ binding enhanced mechanical stability of an archaeal crystallin.

Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and 10FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a “mechanical clamp”. Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the “mechanical clamp” geometry at the termini. M-crystallin is a Ca2+ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and 10FNIII, as well as lacking “mechanical clamp” geometry, can be mechanically resistant. Furthermore, Ca2+ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca2+ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.

Interactions between Trehalose and Amino Acids by Molecular Dynamics Simulations

Although trehalose is used as a protein stabilizer, the mechanism by which this stability is induced is not fully understood at present. In this study, we investigated the interactions between trehalose and all 20 common amino acids using all- atom molecular dynamics simulations. It is found that all the amino acids exhibit a preference for contact with water, especially the polar and charged amino acids.Conversely, only the hydrophobic amino acids were found to have a slight preference for contact with trehalose molecules. This tendency is most pronounced in the case of contact between trehalose and aromatic or hydrophobic side chains, whereas the backbones of each amino acids all show similar propensities for contact with water. Furthermore, hydrogen bonds between amino acids and trehalose were found to be significantly weaker than those between amino acids and water, although both trehalose and water can interact with the amino acids via hydrogen bonds. These findings are important with regard to the exploration of the molecular mechanism of protein stability induced by trehalose and the rational design of highly efficient protein stabilizers.

The Correlation of Expression Levels of HIF-1 and HIF-2 in Hepatocellular Carcinoma with Capsular Invasion, Portal Vein Tumor Thrombi and Patients' Clinical Outcome

The roles of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in the development of hepatocellular carcinoma have not been fully elucidated. Here, we aim to uncover the relationship between the prognosis of hepatocellular carcinoma patients and the expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in tumor tissues. The protein levels of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α were detected by immunohistochemistry on paraffin sections of 126 paired hepatocellular carcinoma tissue and peritumoral tissue samples. The mRNA levels of them were detected by quantitative real-time polymerase chain reaction. High expression of hypoxia-inducible factor-1α was found in 57.1% (72/126) of tumor specimens, compared with 5.6% (7/126) in peritumoral tissues, while high expression of hypoxia-inducible factor-2α was found in only 13.5% (17/126) of tumors, compared with 47.6% (60/126) of peritumoral tissues. There was high expression of hypoxia-inducible factor-1α protein in hepatocellular carcinoma tissues closely associated with capsular infiltration and portal vein invasion, and thus lower overall survival and disease-free survival of hepatocellular carcinoma patients (P < 0.05). No significant association has been found between the expression of hypoxia-inducible factor-2α protein and capsular infiltration, portal vein invasion, overall survival and disease-free survival (P > 0.05). However, patients with high expression of both hypoxia-inducible factor-1α and hypoxia-inducible factor-2α have a significantly worse outcome than patients with low expression of both hypoxia-inducible factor-1α and hypoxia-inducible factor-2α (P < 0.05). The discordant results on expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression and stabilization mechanisms. The association between hypoxia-inducible factor-1α expression and unfavorable outcome indicates the importance of using hypoxia-inducible factor-1α as a treatment target in hepatocellular carcinoma.

Influence of Osmolytes on Protein and Water Structure: A Step To Understanding the Mechanism of Protein Stabilization

Results concerning the thermostability of hen egg white lysozyme in aqueous solutions with stabilizing osmolytes, trimethylamine-N-oxide (TMAO), glycine (Gly), and its N-methyl derivatives, N-methylglycine (NMG), N,N-dimethylglycine (DMG), and N,N,N-trimethylglycine (betaine, TMG), have been presented. The combination of spectroscopic (IR) and calorimetric (DSC) data allowed us to establish a link between osmolytes' influence on water structure and their ability to thermally stabilize protein molecule. Structural and energetic characteristics of stabilizing osmolytes' and lysozyme's hydration water appear to be very similar. The osmolytes increase lysozyme stabilization in the order bulk water < TMAO < TMG < Gly < DMG < NMG, which is consistent with the order corresponding to the value of the most probable oxygen-oxygen distance of water molecules affected by osmolytes in their surrounding. Obtained results verified the hypothesis concerning the role of water molecules in protein stabilization, explained the osmophobic effect, and finally helped to bring us nearer to the exact mechanism of protein stabilization by osmolytes.

Mechanism of Protein Stabilization by Trehalose During Freeze-Drying Analyzed by In Situ Micro-Raman Spectroscopy

Raman investigations were performed in situ during freeze-drying of two model proteins, lysozyme and chymotrypsinogen. The structures of proteins dissolved in 0-30 wt % solutions of trehalose in D2 O were monitored with the fingerprint (800-1800 cm(-1) ) spectrum, simultaneously with freezing, ice sublimation, and water desorption analyzed in the O-D stretching (2200-2700 cm(-1) ) region. In the absence of trehalose, the main changes were detected at the end of primary drying, and correspond to distortion and disordering of secondary structures. A stabilizing effect of trehalose was evidenced in primary and secondary drying stages. Raman images were calculated after freezing and primary drying, providing the distributions of trehalose, water, and protein which occur during the first two stages of the lyophilization cycle. Raman images show a slight heterogeneity in the degree of protein denaturation at the end of primary drying, in relation with the structure of the frozen product observed during freezing. The ability of trehalose to make the protein more rigid was determined as responsible for the protein stabilization during a lyophilization cycle.

Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms

It has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (Koga et al., Nature 2012;491:222-227). By exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (Koga et al., Nature 2012;491:222-227). By using structural databases of proteins isolated from either mesophilic or thermophilic organisms, we here investigate the influence of supersecondary associations on the thermal stability of natural proteins. Our results suggest that β-hairpins of proteins from thermophilic organisms are very frequently characterized by shortenings of the loops. Interestingly, this shortening leads to states that display a very strong preference for the most common connectivity of the strands observed in native protein hairpins. The abundance of selective states in these proteins suggests that they may achieve a high stability by adopting a strategy aimed to reduce the possible conformations of the unfolded ensemble. In this scenario, our data indicate that the shortening is effective if it increases the adherence to these rules. We also show that this mechanism may operate in the stabilization of well-known protein folds (thioredoxin and RNase A). These findings suggest that future investigations aimed at defining mechanism of protein stabilization should also consider these effects.

Temperature stress induces notochord abnormalities and heat shock proteins expression in larval green sturgeon (Acipenser medirostrisAyres 1854)

The effects of thermal stress on survival, development and heat shock protein (hsp) expression of green sturgeon (GS) yolk-sac larvae, from hatching through yolk depletion were investigated to provide insight into effects of highly altered natural river hydrographs. Hatched GS larvae were reared at constant water temperatures 18°C (control) through 28°C at 2°C increments. Larval survival significantly decreased at 26–28°C, with 28°C being lethal. Significant proportions of deformed larvae were found at sub-lethal (20–26°C) and lethal 28°C rearing temperatures, with kyphosis (i.e. backward flexion of notochord) accounting for >99% of morphological deformities. Histological analysis of larvae preparations indicate that elevated water temperature affects notochord cell function and physiology. At rearing temperatures 20–28°C, thermal stress elicited a quick (24 h) and long lasting (yolk-sac absorption) significant over-expression of measured heat shock proteins (hsps), all of which are known components of intracellular protein repair and stabilization mechanism. Thermal sensitivity, as indicated by the incidence of abnormalities and expression of different hsps, varied significantly between crosses. Thermally tolerant progeny exhibited a short but rapid hsp72 (size in kDa) over-expression, and more pronounced hsp60 and hsp90 over-expression, than less tolerant progeny which exhibited a prolonged hsp72 and hsp78 over-expression. At environmentally relevant water temperatures bent larvae exhibited spiral swimming, which in the wild would compromise the ability of emerging larvae to forage, avoid predators, and migrate downstream, ultimately compromising survival and recruitment. Before larvae hsp content can be used as a thermal-stress biomarker for GS, field validation studies are needed.

The micro and macro of RNA function

The central dogma uniquely positions RNA as the key molecular player for translating genetic software into protein hardware. But it has become increasingly clear that RNA also plays an essential regulatory role within cells. MicroRNAs in particular have emerged as ubiquitous and still poorly understood molecules involved in the control of a wide variety of biological processes. RNA has also recently been shown to have important roles in structural assembly within cells. A diverse set of speakers at the Minisymposium on Micro- and Coding RNA highlighted some of these new and often unexpected roles of RNA across a range of length scales. Tracy Johnson (University of California–San Diego) discussed her lab's work on cotranscriptional mRNA splicing. Her talk underscored the importance of ATP-dependent RNA helicases in rearranging RNA secondary structure and the integral role for chromatin modification in coordinating RNA rearrangements with transcription in space and time. Albert Rosana (Owttrim lab, University of Alberta) also discussed RNA helicases within the fascinating context of temperature fluctuations of cyanobacteria. He described his work on the altered expression of the oligomeric RNA helicase CrhR as a temperature stress response. His work provides new insights into RNA regulation, suggesting CrhR autoregulates its expression through a combination of RNA processing and RNA–protein stabilization mechanisms, which in turn leads to temperature-dependent regulation of small regulatory RNAs. Several presentations explored how RNAs play a role in the assembly of large-scale intracellular structures. Magdalena Strzelecka (Heald lab, University of California–Berkeley) discussed her work on RNAs in the assembly of the mitotic spindle. She utilized Xenopus egg extracts and next-generation sequencing approaches to demonstrate a surprising role for splicing factors in mitosis. This suggests the intriguing possibility that RNA processing plays a regulatory role in cell division. Moving to even larger scales, Cliff Brangwynne (Princeton University) discussed his lab's work on the biophysics of ribonucleoprotein (RNP) bodies—large, non–membrane bound assemblies that behave as liquid-phase droplets of RNA and protein. This work exploits the nucleus of Xenopus oocytes to study the nucleolus, an RNP droplet involved in ribosome biogenesis. The presentation revealed some unexpected biophysical consequences of the large length scales involved, with important implications for nuclear RNP organization and function. The nucleolus was also the central theme of a talk by Thoru Pederson (University of Massachusetts Medical School), who summarized his lab's recently published work on a subset of microRNAs localized in the nucleolus of rat myoblasts. He also reported new results on the nucleolar presence of certain mRNAs in these cells, proposing there might be microRNA–mRNA interactions in the nucleolus to pre-set the translational status of exported mRNAs. The remaining talk of the session, by Rebecca Holmes (Tollervey lab, University of Edinburgh), focused on the yeast protein Npl3, a member of the large SR family of RNA-binding proteins with diverse functions in pre-mRNA processing. She described experiments utilizing in vivo cross-linking and deep sequencing, which revealed the binding of Npl3 to a surprising variety of RNA species. Her data suggest an unexpected role for Npl3 in regulating noncoding RNAs (ncRNAs), with consequences for the expression of surrounding genes. These results raise the possibility that other previously described functions of Npl3 may, in fact, be consequences of Npl3 regulation of ncRNAs.

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.

Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency

Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.