Direct evidence for a deprotonated lysine serving as a H-bond "acceptor" in a photoreceptor protein.

Deprotonation or suppression of the pKa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an "acceptor" in a H-bond between the photosensor protein RcaE and its chromophore. Signal splitting and trans-H-bond J coupling observed by NMR spectroscopy provide direct evidence that Lys261 is deprotonated and serves as a H-bond acceptor for the chromophore NH group. Quantum mechanical/molecular mechanical calculations also indicate that this H-bond exists stably. Interestingly, the sidechain amino group of the lysine can act as both donor and acceptor. The remarkable shift in the H-bond characteristics arises from a decrease in solvation, triggered by photoisomerization. Our results provide insights into the dual role of this lysine. This mechanism has broad implications for other biological reactions in which lysine plays a role.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

Efficient identification of compounds suppressing protein precipitation via solvent screening using serial deletion mutants of the target protein.

The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N- or C-terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants. The solubilizing compounds are then identified from the screened solvents. In this study, the C-terminal chemokine receptor-binding region of the cytoplasmic protein, FROUNT (FNT-C), which mediates intracellular signals leading to leukocyte migration, was subjected to the multicomponent screening. In total, 192 solution conditions were tested, using eight terminal deletion mutants of FNT-C. We identified five solvent conditions that solubilized four or five mutants of FNT-C, and the compounds in the screened solvents were then, respectively, assessed in terms of their solubilization ability. The best compound for solubilizing FNT-C was 1,6-hexanediol. Indeed, 1,6-hexanediol bound to FNT-C and suppressed its precipitation, as showed by NMR and dynamic light scattering analyses.

Immediate enamel bond strength of universal adhesives to unground and ground surfaces in different etching modes.

The purpose of this study was to determine the immediate bond effectiveness of universal adhesives to unground and ground enamel surfaces in different etching modes, through shear bond strength (SBS) tests and scanning electron microscopy observations. Three universal adhesives, a conventional two-step self-etch adhesive, and a conventional single-step self-etch adhesive were compared. Human enamel specimens from lower anterior teeth were divided into four groups and subjected to the following treatments: (i) unground enamel in self-etch mode; (ii) ground enamel in self-etch mode; (iii) unground enamel in etch-&-rinse mode; and (iv) ground enamel in etch-&-rinse mode. Bonded assemblies were subjected to SBS testing. All the adhesives showed significantly higher SBS values in etch-&-rinse mode than in self-etch mode, regardless of whether enamel was unground or ground. The influence of the enamel surface condition on SBS was different in different etching modes. Without pre-etching, all tested materials showed lower SBS values in unground enamel than in ground enamel. In etch-&-rinse mode, no significant differences in SBS values were observed between unground enamel and ground enamel for any of the adhesives tested. Phosphoric acid pre-etching before application of self-etch adhesives to an unground enamel surface is essential to enhance initial enamel bond effectiveness.

Perspective: next generation isotope-aided methods for protein NMR spectroscopy.

In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of "TROSY by isotope labeling" has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52-57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.

(13)C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI).

Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14-Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D (13)C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically (13)C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14-Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m c14 and m c38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for (13)C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the (1)H-(13)C signals at the ß-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.

We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and ß-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,ß-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,ß-dihydroxy-α-methylvalerate to α-keto-ß-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Differential Large-Amplitude Breathing Motions in the Interface of FKBP12-Drug Complexes.

The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel.

Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Expression and purification of a GRAS domain of SLR1, the rice DELLA protein.

GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.

Sonographic appearance of the pronator quadratus muscle in healthy volunteers.

OBJECTIVES: The purpose of this study was to investigate the sonographic appearance of the pronator quadratus muscle in healthy volunteers. METHODS: We sonographically evaluated 30 healthy volunteers (15 men and 15 women). The maximum thickness of the pronator quadratus in millimeters on the volar side at the cortical level was measured on sagittal and axial sonographic images with the volunteers' hands supinated. On the axial image, we classified each hand according to the sonographic contour of the pronator quadratus. RESULTS: A significant difference in the maximum pronator quadratus thickness between dominant and nondominant hands was shown on each image (P < .01). A comparison between men and women also showed a significant difference irrespective of image or hand dominance. Approximately 1.5 mm on the sagittal image and 1.4 mm on the axial image seemed to be adequate practical values for detecting pronator quadratus thickening. In terms of the pronator quadratus contour, the convex type appeared in most men in both the dominant and nondominant hands. In women, the convex and concave types appeared often in both the dominant and nondominant hands. CONCLUSIONS: The difference in pronator quadratus thickness between dominant and nondominant hands might need to be taken into account during sonographic studies. However, in many cases, the difference seems to be slight and considerably less than the calculated cutoff value in this study. These results might be useful for sonographic comparison of bilateral pronator quadratus muscles in the clinical setting.

Anteroposterior translation does not correlate with knee flexion after total knee arthroplasty.

BACKGROUND: Stiffness after a TKA can cause patient dissatisfaction and diminished function, therefore it is important to characterize predictors of ROM after TKA. Studies of AP translation in conscious individuals disagree whether AP translation affects maximum knee flexion angle after implantation of a highly congruent sphere and trough geometry PCL-substituting prosthesis in a TKA. QUESTIONS/PURPOSES: We investigated whether AP translation correlated with maximum knee flexion angle (1) in patients who were awake, and (2) who were under anesthesia (to minimize the effects of voluntary muscle contraction) in a TKA with implantation of a PCL-substituting mobile-bearing prosthesis. METHODS: AP translation was examined under both conditions in 34 primary TKAs. Measurements under anesthesia were performed when the patients were having anesthesia for a contralateral TKA. Awake measurements were made within 4 days of that anesthetic session in patients who had no residual sedative effects. The average postoperative interval for the index TKA flexion measurements was 23 months (range, 6-114 months). AP translation was evaluated at 75° flexion using an arthrometer. RESULTS: There was no correlation between postoperative maximum knee flexion and AP translation at 75° during consciousness. There was no correlation between postoperative maximum knee flexion and AP translation under anesthesia. CONCLUSION: AP translation at 75° flexion did not correlate with postoperative maximum knee flexion in either awake or anesthetized patients during a TKA with implantation of a posterior cruciate-substituting prosthesis.

Characteristics and significance of fever during 4 weeks after primary total knee arthroplasty.

PURPOSE: Most previous studies on postoperative fever (POF; ≥38 °C) after total knee arthroplasty (TKA) have reported findings from only the immediate postoperative days (PODs). The hypothesis of the current study is that 4 weeks of follow-up may reveal differences in the characteristics of POF and fever-related factors between a normal inflammatory response and an early acute infection-related response. METHODS: A total of 400 consecutive TKAs (314 patients) were retrospectively investigated. Patients were stratified into those who developed an early acute periprosthetic infection that required subsequent surgical treatment (STG; n = 5 TKAs) and those who did not (non-STG; n = 395 TKAs). RESULTS: Among the 400 knees, 149 (37 %) developed POF, with most reaching a maximum temperature (MT) on POD 0. In 13 TKA patients who had POF with a peak daily temperature ≥38 °C during postoperative weeks 2-4, the causes of POF were respiratory and urinary tract infections (n = 5 for each), superficial infection (n = 2), and periprosthetic infection (n = 1). The STG and non-STG differed significantly with regard to the rate of POF (p = 0.0205) and MT (p = 0.0003), including MTs less than 38 °C, during postoperative weeks 2-4. All five STG patients had elevated C-reactive protein levels and local symptomatic findings before the additional surgery. CONCLUSIONS: The occurrence of POF and MT along with elevated C-reactive protein and local symptomatic findings at 2-4 weeks postoperatively may indicate the need for a positive fever workup to recognize early acute periprosthetic infection.

Length of hospital stay with patient-dependent determination in bilateral scheduled staged total knee arthroplasty.

PURPOSE: The purpose of this study was to approach the length of hospital stay (LOS) by patient-dependent determination and evaluate the effect of contralateral total knee arthroplasty (TKA) on LOS in patients with bilateral osteoarthritis undergoing staged bilateral TKAs. METHODS: One hundred sixty TKAs in 80 patients with bilateral osteoarthritis were evaluated by retrospective review of their medical records. All patients had scheduled staged bilateral TKA. We investigated the length of hospital stay in each TKA and the postoperative day when patients could perform walking and climbing up and down the stairs with one cane independently. Range of motion in each knee was also evaluated in preoperatively and before discharge. The median interval between the first and second surgeries was 12 months. RESULTS: The first and second TKAs showed a median LOS of 37 and 35 days, respectively, with no significant difference (n.s.). The median number of days before independently walking and climbing up and down the stairs with one cane were 8 and 16 after the first TKA and 7 and 15 after the second TKA, respectively, without significant differences (n.s. for walking, n.s. for stairs). CONCLUSIONS: An initial TKA that results in good function might neither facilitate an earlier second rehabilitation nor decrease the LOS. Taking other factors such economics and local conditions that largely influence LOS into account, simultaneous bilateral TKAs might be an effective treatment for bilateral knee arthritis in properly selected patients in terms of decreasing the LOS.

In vivo anteroposterior translation after meniscal-bearing total knee arthroplasty: effects of soft tissue conditions and flexion angle.

PURPOSE: Anteroposterior (AP) joint translation is an important indicator of good clinical outcome following total knee arthroplasty (TKA). This study evaluated the in vivo relationship between changes in the degree of voluntary soft tissue tension and flexion angle versus simultaneous AP translation after TKA. METHODS: A posterior cruciate ligament (PCL)-retaining meniscal-bearing design was investigated in 20 knees of 20 patients. AP translation was measured at 30° and 75° flexion with the KT-2000 arthrometer while patients were anesthetized and non-anesthetized. RESULTS: The mean translations at 30° and 75° were 10.5 and 10.4 mm, respectively, in non-anesthetized patients and 13.8 and 12.7 mm, respectively, in patients under anesthesia. AP translation showed a significant positive correlation with soft tissue tension (p < 0.001), but not with flexion angle (p = 0.366). No interaction was observed between soft tissue tension and the flexion angle in terms of AP translation (p = 0.431). CONCLUSION: Surgeons should recognize that AP translation is greater in anesthetized patients than in non-anesthetized patients, regardless of the flexion angle, with no significant correlation between flexion angle and translation, regardless of the level of consciousness. Because conformity between the tibial insert and femoral component decreases with flexion, whereas the opposing effects of supporting structures, such as muscles, ligaments, and capsules, increases, proper soft tissue tension, particularly retention of a functional PCL, could have an important role in determining AP translation in the current prosthesis design.

Enhancing solution structural analysis of large molecular proteins through optimal stereo array isotope labeling of aromatic amino acids.

The observation of side-chain peaks of aromatic amino acids is the prerequisite for a high-resolution three-dimensional structure determination of proteins by NMR. However, it becomes difficult with increasing molecular size due to an increased transverse relaxation and the control of the relaxation pathway is needed to achieve the observation. We demonstrated that even for the large molecular size of 82 kDa Malate synthase G (MSG), the aromatic 13C-1H (CH) peaks of Tryptophan (Trp) and Phenylalanine (Phe) residues can be observed with high quality using a systematic stable isotope labeling scheme, Stereo-Array Isotope Labeling (SAIL) method. However, the sequence specific assignments of these peaks relied on the use of amino acid substitutions, employing an inefficient method that required many isotopes labeled samples. In this study, we developed novel SAIL amino acids that allow for the observation of the aromatic ring δ,ζ and the aliphatic ß position peak of Phe residues. The application of TROSY-based experiment to the isolated CH moieties resulted in the successful observation of discernible and resolved CH peaks in Phe residues in MSG. In MSG, the sequence-specific assignments of the backbone and Cß positions have already been confirmed. Therefore, using this labeling method, the δ and ß position peaks of Phe residues can be clearly assigned in a sequence-specific and stereospecific manner through experiments based on intra-residue NOE. Furthermore, the NOESY experiment also allows for the acquisition of information pertaining to the conformation of Phe residues, such as the χ1 dihedral angle, providing valuable insights for the determination of accurate protein structures and in dynamic analysis. This new SAIL amino acids open an avenue to achieve a variety of NMR analysis of large molecular proteins, including a high-resolution structure determination and dynamics and interaction analysis.

Radical electron-induced cellulose-semiconductors.

Bio-semiconductors are expected to be similar to organic semiconductors; however, they have not been utilized in application yet. In this study, we show the origin of electron appearance, N- and S-type negative resistances, rectification, and switching effects of semiconductors with energy storage capacities of up to 418.5 mJ/m2 using granulated amorphous kenaf cellulose particles (AKCPs). The radical electrons in AKCP at 295 K appear in cellulose via the glycosidic bond C1-O1·-C4. Hall effect measurements indicate an n-type semiconductor with a carrier concentration of 9.89 × 1015/cm3, which corresponds to a mobility of 10.66 cm2/Vs and an electric resistivity of 9.80 × 102 Ωcm at 298 K. The conduction mechanism in the kenaf tissue was modelled from AC impedance curves. The light and flexible cellulose-semiconductors may open up new avenues in soft electronics such as switching effect devices and bio-sensors, primarily because they are composed of renewable natural compounds.

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids.

The (1)H-(13)C HMQC signals of the (13)CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically (13)CH3-labeled [U-(2)H;(15)N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.

Posterior condylar offset does not correlate with knee flexion after TKA.

BACKGROUND: Studies of medial and lateral femoral posterior condylar offset have disagreed on whether posterior condylar offset affects maximum knee flexion angle after TKA. QUESTIONS/PURPOSES: We asked whether posterior condylar offset was correlated with knee flexion angle 1 year after surgery in (1) a PCL-retaining meniscal-bearing TKA implant, or in (2) a PCL-substituting mobile-bearing TKA implant. METHODS: Knee flexion angle was examined preoperatively and 12 months postoperatively in 170 patients who underwent primary TKAs to clarify the effect of PCL-retaining (85 knees) and PCL-substituting (85 knees) prostheses on knee flexion angle. A quasirandomized design was used; patients were assigned to receive one or the other implant using chart numbers. A quantitative three-dimensional technique with CT was used to examine individual changes in medial and lateral posterior condylar offsets. RESULTS: In PCL-retaining meniscal-bearing knees, there were no significant correlations between posterior condylar offset and knee flexion at 1 year. In these knees, the mean (± SD) postoperative differences in medial and lateral posterior condylar offsets were 0.0 ± 3.6 mm and 3.8 ± 3.6 mm, respectively. The postoperative change in maximum knee flexion angle was -5° ± 15°. In PCL-substituting rotating-platform knees, similarly, there were no significant correlations between posterior condylar offset and knee flexion 1 year after surgery. In these knees, the mean postoperative differences in medial and lateral posterior condylar offsets were -0.5 ± 3.3 mm and 3.3 ± 4.2 mm, respectively. The postoperative change in maximum knee flexion angle was -2° ± 18°. CONCLUSIONS: Differences in individual posterior condylar offset with current PCL-retaining or PCL-substituting prostheses did not correlate with changes in knee flexion 1 year after TKA. We should recognize that correctly identifying which condyle affects the results of the TKA may be difficult with conventional radiographic techniques.