How Visual Expertise Changes Representational Geometry: A Behavioral and Neural Perspective.

Many people develop expertise in specific domains of interest, such as chess, microbiology, radiology, and, the case in point in our study: ornithology. It is poorly understood to what extent such expertise alters brain function. Previous neuroimaging studies of expertise have typically focused upon the category level, for example, selectivity for birds versus nonbird stimuli. We present a multivariate fMRI study focusing upon the representational similarity among objects of expertise at the subordinate level. We compare the neural representational spaces of experts and novices to behavioral judgments. At the behavioral level, ornithologists (n = 20) have more fine-grained and task-dependent representations of item similarity that are more consistent among experts compared to control participants. At the neural level, the neural patterns of item similarity are more distinct and consistent in experts than in novices, which is in line with the behavioral results. In addition, these neural patterns in experts show stronger correlations with behavior compared to novices. These findings were prominent in frontal regions, and some effects were also found in occipitotemporal regions. This study illustrates the potential of an analysis of representational geometry to understand to what extent expertise changes neural information processing.

Is increased activation in the fusiform face area to Greebles a result of appropriate expertise training or caused by Greebles' face likeness?

Background: In 2011, Brants et al. trained eight individuals to become Greeble experts and found neuronal inversion effects [NIEs; i.e., higher fusiform face area (FFA) activity for upright, rather than inverted Greebles]. These effects were also found for faces, both before and after training. By claiming to have replicated the seminal Greeble training study by Gauthier and colleagues in 1999, Brants et al. interpreted these results as participants viewing Greebles as faces throughout training, contrary to the original argument of subjects becoming Greeble experts only after training. However, Brants et al.'s claim presents two issues. First, their behavioral training results did not replicate those of Gauthier and Tarr conducted in 1997 and 1998, raising concerns of whether the right training regime had been adopted. Second, both a literature review and meta-analysis of NIEs in the FFA suggest its impotency as an index of the face(-like) processing. Objectives: To empirically evaluate these issues, the present study compared two documented training paradigms Gauthier and colleagues in 1997 and 1998, and compared their impact on the brain. Methods: Sixteen NCKU undergraduate and graduate students (nine girls) were recruited. Sixty Greeble exemplars were categorized by two genders, five families, and six individual levels. The participants were randomly divided into two groups (one for Greeble classification at all three levels and the other for gender- and individual-level training). Several fMRI tasks were administered at various time points, specifically, before training (1st), during training (2nd), and typically no <24 h after reaching expertise criterion (3rd). Results: The ROI analysis results showed significant increases in the FFA for Greebles, and a clear neural "adaptation," both only in the Gauthier97 group and only after training, reflecting clear modulation of extensive experiences following an "appropriate" training regime. In both groups, no clear NIEs for faces nor Greebles were found, which was also in line with the review of extant studies bearing this comparison. Conclusion: Collectively, these results invalidate the assumptions behind Brants et al.'s findings.

Perceptual Learning with Complex Objects: A Comparison between Full-Practice Training and Memory Reactivation.

Perception improves with repeated exposure. Evidence has shown object recognition can be improved by training for multiple days in adults. Recently, a study of Amar-Halpert et al. (2017) has compared the learning effect of repetitive and brief, at-threshold training on a discrimination task and reported similar improvement in both groups. The finding is interpreted as evidence that memory reactivation benefits discrimination learning. This raises the question how this process might influence different perceptual tasks, including tasks with more complex visual stimuli. Here, this preregistered study investigates whether reactivation induces improvements in a visual object learning task that includes more complex visual stimuli. Participants were trained to recognize a set of objects during 5 d of training. After the initial training, a group was trained with repeated practice, the other a few near-threshold reactivation trials. In both groups, we found improved object recognition at brief exposure durations. Traditional intense training shows a daily improvement; however, the group with reactivation does not reach the same level of improvement. Our findings show that reactivation has a smaller effect relative to large amounts of practice.

A brain network that supports consensus-seeking and conflict-resolving of college couples' shopping interaction.

One of the typical campus scenes is the social interaction between college couples, and the lesson couples must keep learning is to adapt to each other. This fMRI study investigated the shopping interactions of 30 college couples, one lying inside and the other outside the scanner, beholding the same item from two connected PCs, making preference ratings and subsequent buy/not-buy decisions. The behavioral results showed the clear modulation of significant others' preferences onto one's own decisions, and the contrast of the "shop-together vs. shop-alone", and the "congruent (both liked or disliked the item, 68%) vs. incongruent (one liked but the other disliked, and vice versa)" together trials, both revealed bilateral temporal parietal junction (TPJ) among other reward-related regions, likely reflecting mentalizing during preference harmony. Moreover, when contrasting "own-high/other-low vs. own-low/other-high" incongruent trials, left anterior inferior parietal lobule (l-aIPL) was parametrically mapped, and the "yield (e.g., own-high/not-buy) vs. insist (e.g., own-low/not-buy)" modulation further revealed left lateral-IPL (l-lIPL), together with left TPJ forming a local social decision network that was further constrained by the mediation analysis among left TPJ-lIPL-aIPL. In sum, these results exemplify, via the two-person fMRI, the neural substrate of shopping interactions between couples.

Structural Insight into Integrin Recognition and Anticancer Activity of Echistatin.

Echistatin (Ech) is a short disintegrin with a long 42NPHKGPAT C-terminal tail. We determined the 3-D structure of Ech by X-ray crystallography. Superimposition of the structures of chains A and B showed conformational differences in their RGD loops and C-termini. The chain A structure is consistent with our NMR analysis that the GPAT residues of the C-terminus cannot be observed due to high flexibility. The hydrogen bond patterns of the RGD loop and between the RGD loop and C-terminus in Ech were the same as those of the corresponding residues in medium disintegrins. The mutant with C-terminal HKGPAT truncation caused 6.4-, 7.0-, 11.7-, and 18.6-fold decreases in inhibiting integrins αvß3, αIIbß3, αvß5, and α5ß1. Mutagenesis of the C-terminus showed that the H44A mutant caused 2.5- and 4.4-fold increases in inhibiting αIIbß3 and α5ß1, and the K45A mutant caused a 2.6-fold decrease in inhibiting αIIbß3. We found that Ech inhibited VEGF-induced HUVEC proliferation with an IC50 value of 103.2 nM and inhibited the migration of A375, U373MG, and Panc-1 tumor cells with IC50 values of 1.5, 5.7, and 154.5 nM. These findings suggest that Ech is a potential anticancer agent, and its C-terminal region can be optimized to improve its anticancer activity.

Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin.

Rhodostomin (Rho) is a snake venom protein containing an RGD motif that specifically inhibits the integrin-binding function. Rho produced in Pichia pastoris inhibits platelet aggregation with a K(I) of 78 nM as potent as native Rho. In contrast, its D51E mutant inhibits platelet aggregation with a K(I) of 49 muM. Structural analysis of Rho and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets. There are no structural backbone differences between the RG[D/E] loop which extends outward from the protein core and the RG[D/E] sequence at its apex in a four-residue RG[D/E]M type I turn. Two minor differences between Rho and its D51E mutant were only found from their backbone dynamics and 3D structures. The R(2) value of E51 is 13% higher than that of the D51 residue. A difference in the charge separation of 1.76 A was found between the sidechains of positive (R49) and negative residues (D51 or E51).The docking of Rho into integrin alphavbeta3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho were formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex. Our findings suggest that the interactions between both the sidechain and backbone of the D residue of RGD-containing ligands and integrin are important for their binding.

Determinants of peripheral arterial stiffness in patients with chronic kidney disease in southern Taiwan.

High prevalences of peripheral artery occlusive disease (PAOD) and increased arterial stiffness have been reported in patients with chronic kidney disease (CKD). However, these have not been assessed in Taiwan where the prevalence of CKD is high. The aim of this study was to investigate the determinants of PAOD and arterial stiffness in patients with CKD in southern Taiwan. We enrolled 169 patients with stage 3-5 CKD in one regional hospital. Ankle-brachial index (ABI) and brachial-ankle pulse wave velocity were measured using an ABI-form device (Colin VP1000). In multivariate analysis, ABI<0.9 was positively correlated with the presence of diabetes mellitus (p=0.014) and negatively correlated with the estimated glomerular filtration rate (eGFR) (p=0.049), and increased brachial-ankle pulse wave velocity was correlated with increased age, diabetes mellitus, increased systolic blood pressure, decreased pulse pressure and decreased eGFR. This study identified determinants of PAOD and arterial stiffness in patients with CKD in one hospital in southern Taiwan. In addition to the traditional atherosclerotic risk factors, decreased eGFR was also correlated with PAOD and increased arterial stiffness in these patients.

Increased Aortic Arch Calcification and Cardiomegaly is Associated with Rapid Renal Progression and Increased Cardiovascular Mortality in Chronic Kidney Disease.

Vascular calcification and cardiomegaly are highly prevalent in chronic kidney disease (CKD) patients. However, the association of the combination of aortic arch calcification (AoAC) and cardio-thoracic ratio (CTR) with clinical outcomes in patients with CKD is not well investigated. This study investigated whether the combination of AoAC and CTR is associated with poor clinical outcomes in CKD stages 3-5 patients. We enrolled 568 CKD patients, and AoAC and CTR were determined by chest radiography at enrollment. Rapid renal progression was defined as estimated glomerular filtration rate (eGFR) decline over 3 ml/min/1.73 m2 per year. Both AoAC score and CTR were significantly associated with rapid renal progression. High CTR was correlated with increased risk for cardiovascular mortality. We stratified the patients into four groups according to the median AoAC score of 4 and CTR of 50%. Those with AoAC ≥ 4 and CTR ≥ 50% (vs. AoAC score < 4 and CTR < 50%) were associated with eGFR decline over 3 ml/min/1.73 m2/year and cardiovascular mortality. AoAC and CTR were independently associated with eGFR slope. In conclusion, the combination of increased AoAC and cardiomegaly was associated with rapid renal progression and increased cardiovascular mortality in patients with CKD stage 3-5 patients. We suggest that evaluating AoAC and CTR on chest plain radiography may be a simple and inexpensive method for detecting CKD patients at high risk for adverse clinical outcomes.

Associated risk factors for abnormal ankle-brachial index in hemodialysis patients in a hospital.

Ankle-brachial index (ABI) is a marker for peripheral artery disease and can predict mortality in hemodialysis patients. However, it is seldom studied in southern Taiwan, an area with high prevalence of end-stage renal disease (ESRD). The aim of this study was to investigate the prevalence and associated risk factors for peripheral artery disease in the ESRD population in a hospital. All routine hemodialysis patients in one regional hospital were included except for six patients who refused ABI examinations and four patients with atrial fibrillation. Finally, 225 patients formed our study group. ABI was measured using an ABI-form device (Colin VP1000). The prevalence of ABI < 0.9 and > or = 1.3 was 15.6% and 5.8%, respectively. ABI < 0.9 was independently associated with advanced age (p = 0.027), increased pulse pressure (p = 0.005), increased hematocrit (p = 0.008) and decreased serum albumin level (p = 0.009). In addition, ABI > or = 1.3 was significantly associated with diabetes mellitus (p = 0.019). This study demonstrated the associated risk factors of peripheral artery disease in patients with hemodialysis in a hospital. ESRD patients of advanced age and with increased pulse pressure, increased hematocrit and decreased serum albumin level had a relatively high risk for ABI < 0.9 and patients with diabetes had a relatively high risk for ABI > or = 1.3.

Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbß3 recognition.

Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbß3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbß3 > αvß3 ≥ α5ß1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbß3 mutant. The 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-65PRYH and 48ARGDWN-65PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48ARGDWN-65PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbß3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin αIIbß3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-π interaction with the ß3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.

Streptococcal pyrogenic exotoxin B inhibits apoptotic cell clearance by macrophages through protein S cleavage.

Clearance of apoptotic cells by macrophages plays an important role in maintaining tissue homeostasis. Previous study indicated that streptococcal pyrogenic exotoxin B (SPE B) reduces phagocytic activity in group A streptococcus (GAS) infection. Here, we demonstrate that SPE B causes an inhibitory effect on protein S-mediated phagocytosis. In the presence of SPE B, serum- and purified protein S-mediated phagocytosis of apoptotic cells were significantly inhibited. The binding abilities of protein S to apoptotic cells were decreased by treatment with SPE B. Bacterial culture supernatants from GAS NZ131 strain also caused a reduction of protein S binding to apoptotic cells, but speB mutant strain did not. SPE B directly cleaved protein S in vitro and in vivo, whereas a lower level of cleavage occurred in mice infected with a speB isogenic mutant strain. SPE B-mediated initial cleavage of protein S caused a disruption of phagocytosis, and also resulted in a loss of binding ability of protein S-associated C4b-binding protein to apoptotic cells. Taken together, these results suggest a novel pathogenic role of SPE B that initiates protein S degradation followed by the inhibition of apoptotic cell clearance by macrophages.

Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.

Effect of P to A mutation of the N-terminal residue adjacent to the Rgd motif on rhodostomin: importance of dynamics in integrin recognition.

Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4-11.5 times more actively inhibited integrin α5ß1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5ß1. However, the backbone dynamics of RGD residues were different. The values of the R(2) relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S(2), R(ex), and τ(e). The S(2) values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The R(ex) values of Rho residues R49 and D51 were 0.91 s(-1) and 1.42 s(-1); however, no R(ex) was found for those of the P48A mutant. The τ(e) values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5ß1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5ß1.

Dynamics and functional differences between dendroaspin and rhodostomin: insights into protein scaffolds in integrin recognition.

Dendroaspin (Den) and rhodostomin (Rho) are snake venom proteins containing a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris. The recombinant Den and Rho inhibited platelet aggregation with the K(I) values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin αIIbß3 and 2.5 times less active when inhibiting the integrin αvß3. In contrast, Den and Rho were similarly active when inhibiting the integrin α5ß1 with the IC50 values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl-glycyl-aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin αvß3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif.

Solution structure and backbone dynamics of the DNA-binding domain of FOXP1: insight into its domain swapping and DNA binding.

FOXP1 belongs to the P-subfamily of forkhead transcription factors and contains a conserved forkhead DNA-binding domain. According to size exclusion chromatography analysis, the forkhead domain of FOXP1 existed as a mixture of monomer and dimer. The dissociation constants of the forkhead domain of wild-type, C61S, and C61Y mutants of FOXP1 were 27.3, 28.8, and 332.0 µM, respectively. In contrast, FOXP1 A39P mutant formed only a monomer. NMR analysis also showed that FOXP1 C61S and C61Y mutants existed as a mixture. The solution structure of FOXP1 A39P/C61Y mutant was similar to the X-ray structure of the FOXP2 monomer. Comparison of backbone dynamics of FOXP1 A39P/C61Y and C61Y mutants showed that the residues preceding helix 3, the hinge region, exhibited the largest conformational exchange in FOXP1 monomer. The A39 residue of FOXP1 dimer has a lower order parameter with internal motion on the ps-ns timescale, suggesting that the dynamics of the hinge region of FOXP1 are important in the formation of the swapped dimer. The analysis also showed that the residues exhibiting the motions on the ps-ns and µs-ms timescales were located at the DNA-binding surface of FOXP1, suggesting the interactions between FOXP1 and DNA may be highly dynamic.

Hyperuricemia as an independent risk factor of chronic kidney disease in middle-aged and elderly population.

INTRODUCTION: Hyperuricemia in the general population remains controversial, in terms of it being considered a risk factor for chronic kidney disease (CKD). Within this context, we evaluated the effects of hyperuricemia on renal function in older Taiwanese adults. METHODS: From January 2002 to December 2006, we conducted a community-based medical screening program involving 31,331 subjects older than 40 years. According to the National Kidney Foundation guidelines, stage 3 to 5 patients with CKD were included for analysis. Age, body mass index, systolic blood pressure, fasting plasma glucose, triglyceride, cholesterol and proteinuria were considered potential confounders. RESULTS: Participants with hyperuricemia tended to have higher systolic blood pressure, sugar levels, body mass index, and cholesterol and triglyceride levels but lower estimated glomerular filtration rate (eGFR) levels; eGFR negatively correlated with serum uric acid level. By using multiple logistic regression models before and after adjusting for any confounding factors, we noted that participants with hyperuricemia had a 4.036-fold (odds ratios = 4.036) and 3.649-fold (odds ratios = 3.649) increased risk for CKD, respectively, compared with the control group. We used multiple linear regression analysis to examine the association of serum uric acid level and eGFR at different stages of CKD; significance was found only in participants with stage 3 CKD and not in participants with stages 4 or 5. CONCLUSIONS: Hyperuricemia is an independent risk factor for CKD in middle-aged and elderly Taiwanese adults. Thus, an effective screening program that identifies people with hyperuricemia is warranted.

Hypertriglyceridemia: an independent risk factor of chronic kidney disease in Taiwanese adults.

BACKGROUND: The prevalence and incidence of chronic kidney disease (CKD) are relatively high in Taiwanese patients than in patients of other countries, particularly in the older age groups. Dyslipidemia in patients with CKD has been recognized as a risk factor for disease progression but the role of triglycerides (TGs) remains controversial. With this regard, we evaluated the effects of hypertriglyceridemia on renal function in Taiwanese adults (aged >or=40 years). METHODS: From January 2002 to December 2006, we conducted a community-based medical screening program in Chiayi County with 18,422 subjects (aged >or=40 years). The CKD was defined as an estimated glomerular filtration rate of <60 mL min 1.73 m. Age, body mass index, systolic blood pressure, fasting plasma glucose, and serum total cholesterol were considered as potential confounders. RESULT: The CKD was prevalent in 24.2% of the middle-aged and elderly population. By using multiple logistic regression models, we determined that old age and elevated levels of body mass index, systolic blood pressure, fasting plasma glucose, and cholesterol were associated with CKD. The adjusted odds ratios of CKD in participants with serum TG >==200 mg/dL was 1.901 (95% confidence interval: 1.07-3.36; P < 0.05) and in participants with serum TG > 500 mg/dL it increased to 2.205 (1.33-3.64, P < 0.05). CONCLUSION: Hypertriglyceridemia is an independent risk factor for CKD in Taiwanese adults. Thus, an effective screening program that identifies people with hypertriglyceridemia is warranted.

Expression and characterization of recombinant human cytochrome c in E. coli.

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we expressed human cytochrome c in E. coli and purified it to homogeneity with a yield of 10-15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. On the basis of our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide evidence that human cytochrome c expressed in E. coli possesses a similar function and structure to that of the horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.

Maturation processing and characterization of streptopain.

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.