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RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.

Connexin family proteins assemble into hexameric hemichannels in a cell membrane, which dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). The most ubiquitously expressed connexin Cx43 plays important roles in numerous biological processes. Here we report cryo-EM structures of Cx43 GJIChs at 3.1–3.6 Å resolutions, which show dynamic conformational changes of N-terminal helices (NTHs) caused by pH change or C-terminal truncation. Cx43 GJIChs in a channel-closing condition contain 12 protomers in gate-covering NTH (GCN) conformation, while those in opening conditions have varying compositions of GCNs and pore-lining NTHs (PLNs) resulting in various pore dimensions and electrostatic surface potentials. GCN-to-PLN transition accompanies π-helix formation in the first transmembrane helix (TM1), movement of TM2-4 that creates a side opening to the membrane, and structural stabilization of the cytoplasmic loop. Our study provides structural insights into the intercellular ion/metabolite transfer and the lateral lipid transport through Cx43 GJICh.

The production of biomolecules that require human‐specific lipid environments is extremely useful for basic research and medical applications. In article number 2000154, Seong‐Jun Kim, Jae‐Sung Woo, Sangsu Bae, and co‐workers integrate multiple proteins or virus antigens into defined transcriptional hotspots in the human genome via a homology‐independent targeted insertion method using CRISPR nucleases. This system is similar to a production pipeline of biomolecules in a factory controlled by CRISPR.

We report a novel high-throughput screening tool for drug discovery of direct-acting antiviral agents against RNA viruses., Direct-acting agents against viral components are considered as the most promising candidates for the successful antiviral therapeutics. To date, no direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases. RNA-dependent RNA polymerase (RdRp), an RNA virus–specific enzyme highly conserved among various viral families, has been known as the broad-range antiviral drug target. Here, we developed an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity. We used the RANGO system to a high-throughput chemical screening to identify novel direct-acting antiviral drug candidates targeting DV RdRp from the FDA-approved small-molecule library. RANGO accelerated the massive selection of drug candidates. We found that one of the selected hit compounds, montelukast, showed antiviral activity in vitro and in vivo by directly inhibiting replication of DV and thus relieved related symptoms.

RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

β-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of β-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the β-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of β-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important β-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of β-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against β-lactam-resistant PBPs.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 A resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 Å resolution, respectively, and compared with previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a second-site suppressor.

N HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediateanalog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active siteregion, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while theyexperienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similarto the active site of KSI as described by the crystal structure. These observations indicate that a combined useof paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site ofKSI, and can be applied to the analysis of both active site and function in unknown protein.Key Words : NMR, Ketosteroid isomerase, Active site, Paramagnetic agent, EquileninIntroductionIt has been known that the protein active site plays asignificant role in various physiological processes. Thecatalytic process is a binding reaction of protein similar to alock and key model where the substrate behaves like a keyfitting in the lock by interaction between substrate andresidues of active site in the protein. The active site of theprotein fits to the substrate conformation in size, shape,charge, and hydrophobic or hydrophilic character, or a littleconformational change is induced in order to ensure precisebinding to the substrate.

Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.

AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P

YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 angstrom resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa.

The members of the ARM/HEAT repeat-containing protein superfamily in eukaryotes have been known to mediate protein-protein interactions by using their concave surface. However, little is known about the ARM/HEAT repeat proteins in prokaryotes. Here we report the crystal structure of TON1937, a hypothetical protein from the hyperthermophilic archaeon Thermococcus onnurineus NA1. The structure reveals a crescent-shaped molecule composed of a double layer of α-helices with seven anti-parallel α-helical repeats. A structure-based sequence alignment of the α-helical repeats identified a conserved pattern of hydrophobic or aliphatic residues reminiscent of the consensus sequence of eukaryotic HEAT repeats. The individual repeats of TON1937 also share high structural similarity with the canonical eukaryotic HEAT repeats. In addition, the concave surface of TON1937 is proposed to be its potential binding interface based on this structural comparison and its surface properties. These observations lead us to speculate that the archaeal HEAT-like repeats of TON1937 have evolved to engage in protein-protein interactions in the same manner as eukaryotic HEAT repeats.

Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and structural integrity of hGH. Thus, the nanoporous release device could be a potential delivery system for the long-term controlled release of therapeutic proteins fused with the hybrid Fc fragment.

Objective The aim of this study was to identify apoptosis-related genes of ovarian cancer cell lines following cisplatin treatment. Methods We used IC(50) values and fluorescence-activated cell sorting analysis to compare cell death in 2 ovarian cancer cell lines, namely, SKOV-3 and OVCAR-3, upon treatment with cisplatin. Moreover, the change in transcriptional levels of apoptosis-associated genes was measured with a dendron-modified DNA microarray. Results The protein levels for the up-regulated genes in each cell line were validated to identify the molecules that may determine the cellular behavior of cisplatin resistance. Eight genes were over-expressed in the 2 cell lines. The cisplatin-induced up-regulation of DAD1 in transcriptional and protein levels contributed to the cisplatin resistance of OVCAR-3, and the up-regulation of FASTK and TNFRSF11A in SKOV-3 resulted in its higher sensitivity to cisplatin than that of OVCAR-3. Conclusion In the present study, we have identified a set of genes responsible for apoptosis following cisplatin treatment in ovarian cancer cell lines. These genes may give information about the understanding of cisplatin-induced apoptosis in ovarian cancer.

MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule.

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

We used xenon-perturbed 1H-15N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the beta3-, beta5- and beta6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7Å in 0M urea, 17.3Å in 5.2M urea, and 25.1Å in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using 1H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.

KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.