Lys Groups Research Articles

Optimal dietary lysine improves the growth performance, molting frequency, lipid metabolism, immunity and antioxidant of postlarval mud crab Scylla paramamosain

A 56-day feeding trial was designed to investigate the effect of different dietary lysine (Lys) levels on the growth, molting, lipid metabolism, antioxidant, and immunity of postlarva Scylla paramosain. Six diets with 1.20%, 1.46%, 1.84%, 2.06%, 2.35%, and 2.67% Lys levels (dry matter) were formulated and experimented on the postlarva Scylla paramamosain. The results showed that Scylla paramamosain fed with the 2.06% lysine obtained the highest growth performance and the highest molting frequency. Appropriate dietary Lys levels (1.73–2.03%) can improve the antioxidant capacity of Scylla paramamosain. Although unaffected by dietary Lys, the whole-body lipid content of the 1.20% Lys group was numerically higher than those of the 1.46–2.67% Lys groups. The variation in whole-body crude protein content showed a similar trend with those in the content of EAA and NEAAs, with the highest whole-body crude protein content appearing in the 2.06% Lys group. The crabs obtained the highest expression of igf, fas, EcR and fabp genes in the 2.06% Lys group. When dietary Lys levels were > 1.20%, the transcription level of immune related-genes increased. As suggested by the results of the second order polynomial regression analysis of weight gain, the Lys requirement of postlarva Scylla paramamosain is 2.03% (4.51% of dietary protein).

L-Lysine Ameliorates Diabetic Nephropathy in Rats with Streptozotocin-Induced Diabetes Mellitus.

Introduction Diabetic nephropathy is one of the leading causes of end-stage renal disease worldwide. Uncontrolled hyperglycemia and subsequent production of glycation end-products activate the paths which lead to diabetic nephropathy. The aim of this study was to assess the effects of L-lysine on antioxidant capacity, biochemical factors, kidney function, HSP70 level, and the expression of the TGFβ, VEGF, and RAGE genes in rats with streptozocin-induced diabetes mellitus. Methods Thirty-two male Wistar rats were randomly allocated to four eight-rat groups, namely, a healthy group, a diabetic group treated with vehicle (DM + vehicle), a diabetic group treated with L-lysine (DM + Lys), and a healthy group treated with L-lysine (healthy + Lys). Rats in the DM + Lys and the healthy + Lys groups were treated with L-lysine 0.15%. The levels of fasting blood glucose, insulin, HbA1C, advanced glycation end-products (AGEs), lipid profile, serum creatinine, blood urea nitrogen, glomerular filtration rate, urine microalbumin, oxidative stress parameters, kidney histology and morphology, and TGFβ, VEGF, and RAGE gene expressions were assessed. Findings. An eight-week treatment with L-lysine significantly reduced the levels of fasting blood glucose, AGEs, kidney function parameters, oxidative stress parameters, lipid profile, and the TGFβ, VEGF, and RAGE gene expression and significantly increased the levels of serum insulin and tissue HSP70. Conclusion Treatment with L-lysine seems to slow down the progression of diabetic nephropathy.

Meranzin Hydrate Improves Depression-Like Behaviors and Hypomotility via Ghrelin and Neurocircuitry.

To investigate whether meranzin hydrate (MH) can alleviate depression-like behavior and hypomotility similar to Chaihu Shugan Powder (CSP), and further explore the potential common mechanisms. Totally 120 Spraque-Dawley rats were randomly divided into 5-8 groups including sham, vehicle, fluoxetine (20 mg/kg), mosapride (10 mg/kg), CSP (30 g/kg), MH (9.18 mg/kg), [D-Lys3]-GHRP-6 (Dlys, 0.5 mg/kg), and MH+Dlys groups by a random number table, 8 rats in each group. And 32 mice were randomly divided into wild-type, MH (18 mg/kg), growth hormone secretagogue receptor-knockout (GHSR-KO), and GHSR+MH groups, 8 mice in each group. The forced swimming test (FST), open field test (OFT), tail suspension test (TST), gastric emptying (GE) test, and intestinal transit (IT) test were used to assess antidepressant and prokinetic (AP) effects after drug single administration for 30 min with absorbable identification in rats and mice, respectively. The protein expression levels of brain-derived neurotrophic factor (BDNF) and phosphorylated mammalian target of rapamycin (p-mTOR) in the hippocampus of rats were evaluated by Western blot. The differences in functional brain changes were determined via 7.0 T functional magnetic resonance imaging-blood oxygen level-dependent (fMRI-BOLD). MH treatment improved depression-like behavior (FST, OFT) and hypomotility (GE, IT) in the acute forced swimming (FS) rats (all P<0.05), and the effects are similar to the parent formula CSP. The ghrelin antagonist [D-Lys3]-GHRP-6 inhibited the effect of MH on FST and GE (P<0.05). Similarly, MH treatment also alleviated depression-like behavior (FST, TST) in the wild-type mice, however, no effects were found in the GHSR KO mice. Additionally, administration of MH significantly stimulated BDNF and p-mTOR protein expressions in the hippocampus (both P<0.01), which were also prevented by [D-Lys3]-GHRP-6 (P<0.01). Besides, 3 main BOLD foci following acute FS rats implicated activity in hippocampus-thalamus-basal ganglia (HTB) circuits. The [D-Lys3]-GHRP-6 synchronously inhibited BOLD HTB foci. As expected, prokinetic mosapride only had effects on the thalamus and basal ganglia, but not on the hippocampus. Within the HTB, the hippocampus is implicated in depression and FD. MH accounts for part of AP effects of parent formula CSP in acute FS rats, mainly via ghrelin-related shared regulation coupled to BOLD signals in brain areas. This novel functionally connection of HTB following acute stress, treatment, and regulation highlights anti-depression unified theory.

Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads.

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.

Restriction of dietary digestible lysine allowance in grower phase reduces the occurrence of white striping in broiler chickens

Dietary lysine (Lys) or metabolizable energy (ME) and amino acid (AA) density were reduced during different growth phases to prevent the occurrence of white striping (WS) in broiler chickens. The study consisted of a 49-day rearing experiment involving a total of 390 one-day-old Ross 308 male broiler chickens randomly distributed into 5 experimental groups comprising of 30 replicate pens (13 chickens/pen). One group served as control group fed diets according to the nutrient requirements. Two groups were fed 15 % low digestible lysine (dLys) in grower (GRO low Lys) or grower and finisher diets (GF low Lys). Other groups received 5% low ME and AA density in grower (GRO low ME AA) or grower and finisher diets (GF low ME AA). Lowering dLys suppressed the growth performance during the respective growth phases. BW gain was lower in GF low Lys group than control group (P = 0.010). Broilers in GRO low Lys and GF low Lys groups had lower FI at d 0−49 than those in control group (P = 0.006). Birds in control, GRO low Lys, and GRO low ME AA groups showed better FCR (P < 0.001). Slaughter weight was lower in GF low Lys group than other groups (P = 0.003). Carcass yield was lower in GRO low Lys and GF low Lys groups in comparison with control group (P = 0.004). Breast yield was lower in GF low Lys group whereas broilers fed GRO low Lys diets had lower breast yield than those in control group (P < 0.001). Meat quality was similar among the treatments except drip loss that was lowest in broilers in control group compared with other groups (P = 0.001). Crude protein increased (P = 0.045) whereas crude fat content of breast meat decreased (P = 0.006) in broiler chickens fed low dLys diets. Feeding low dLys diets reduced the incidence and severity of WS compared with other groups (P < 0.001). It is concluded that reducing the dietary dLys levels during grower phase reduced the development of WS in broiler chickens without affecting the overall growth performance.

Cross-linked enzyme lyophilisates (CLELs) of urease: A new method to immobilize ureases

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.

Dietary Supplementation with Lysine and Threonine Modulates the Performance and Plasma Metabolites of Broiler Chicken.

Here, we investigated whether the optimal threonine (Thr) to lysine (Lys) ratio in high Lys diet improves the growth performance of modern broiler chickens at finisher period and determined the possible mechanism underlying improvement in the growth performance of chickens fed with high Lys or Lys + Thr diet using metabolome analyses. Eighteen 21-day-old chickens housed in individual cages were randomly divided into three groups of six chickens fed with different diets as follows: control diet, high Lys diet (150% Lys content of National Research Council requirement), and high Lys + Thr diet (0.68 of Thr/Lys in high Lys diet). Body weight gain (BWG) increased in chickens receiving high Lys diet as compared with those fed with the control diet (P<0.05); no significant difference was observed in BWG of chickens from high Lys + Thr and high Lys groups. Feed conversion ratio (FCR) was lower in chickens fed with high Lys or high Lys + Thr diet than in those on the control diet. Serotonin concentration increased in the plasma of chickens fed with high Lys diet as compared to those fed with other diets. A negative correlation was observed between plasma serotonin concentration and FCR. These results provide the first evidence on the use of high Lys in broiler diets to reduce FCR during finisher period, which may be associated with change in plasma serotonin concentration. These findings suggest that high Lys content in finisher diet, but not high Thr + Lys diet, may affect the peripheral serotonergic metabolism and improve FCR. Thus, plasma serotonin may serve as a biomarker of FCR in broilers.

Quaternary Structure Analysis of a Hemoglobin Core in Hemoglobin-Albumin Cluster.

A core-shell ensemble of bovine hemoglobin (Hb) and human serum albumin (HSA) is an artificial O2 carrier as a red blood cell substitute. This protein particle is created by covalent wrapping of a carbonyl Hb with HSAs: HbR-HSA 3 cluster, where HbR signifies the use of carbonyl Hb (relaxed (R) state conformation) as a starting material. The HbR-HSA 3 cluster exhibits high O2 affinity and low cooperativity. Analysis of the quaternary structure of the central HbR in the cluster revealed that its high O2 affinity is attributed to the physically immobile HbR nucleus. Circular dichroism and UV-vis absorption spectroscopy showed that the structure of deoxy HbR core closely resembles the R-state. The crystal structure of Lys-modified carbonyl HbR was superimposed on that of carbonyl Hb. These results imply that chemical modifications of the surface Lys groups and Cys-93(β) of the carbonyl Hb with cross-linking agent interfered in the quaternary structure movement from the R-state to tense (T) state. As expected, coupling of deoxy Hb (T-state) with HSAs yielded HbT-HSA 3 cluster having low O2 affinity. The mixing of HbR-HSA 3 and HbT-HSA 3 clusters conferred a tailor-made formulation of artificial O2 carrier with a desired O2 affinity ( P50).

Dietary L-lysine supplementation altered the content of pancreatic polypeptide, enzymes involved in glutamine metabolism, and β-actin in rats.

This study investigated the effects of Lys supplementation on serum pancreatic polypeptide (PP), glutamine (Gln) levels and the expression of PP, Gln synthetase (GlnS), glutaminase (Gls) and β-actin in different tissues such as pancreas, skeletal muscle, liver and kidney in rats. Male Sprague-Dawley rats were fed diets containing 7% casein supplemented with either 0% (Control), 1%, 1.5%, 3% Lys or 3% Lys with 1.5% Arg for a week. All rats were necropsied for collection of blood and tissues. Expression of PP, GlnS, Gls, and β-actin in tissues were determined using Western blotting. The results showed that the rats fed 3% supplemental Lys had significantly lower body weight gain (BWG) and food intake than the ≤ 1.5% Lys groups (P < 0.05). Supplementation with ≥ 1% Lys increased serum PP level (P < 0.05), but had no significant effect on pancreatic PP abundance (P > 0.05). GlnS expression was significantly lowered in skeletal muscle by ≥ 1.5% supplemental Lys compared to the Control (P < 0.05). The expression of Gls in the kidney was increased by the addition of 1.5% Arg to 3% Lys diet (P < 0.05). Liver β-actin significantly increased with both Lys and Arg supplementation and muscle β-actin significantly decreased (P < 0.05) with ≥ 1.5% supplemental Lys. Kidney β-actin significantly increased with Arg supplementation vs 3% Lys alone (P < 0.05). These results showed that dietary supplementation with ≥ 1.5% Lys significantly suppressed GlnS expression in the skeletal muscle, which may contribute to the decreased serum Gln levels, and that increased serum PP by Lys may be due to suppressed catabolism rather than increased synthesis of PP. Lys-induced PP may play a role in reducing food intake and BWG.

Loop electrostatics modulates the intersubunit interactions in ferritin.

Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.

The Influence of Dietary Lysine on Yellow Perch Female Reproductive Performance and the Quality of Eggs

AbstractThe objectives of this study were to (1) evaluate if dietary lysine (Lys) has an effect on the free amino acid (FAA) pool of Yellow Perch Perca flavescens eggs, and (2) determine how dietary Lys influences the reproductive performance and eggs fertilization rate (embryo survival) of female Yellow Perch. Two‐year‐old Yellow Perch of initial size of approximately 75 g were randomly distributed into six 400‐L tanks at 32±1 fish per tank. This experiment included two wheat gluten‐based diets in triplicate Lys‐deficient ([−]Lys) and Lys‐supplemented ([+]Lys; 2.23% in dry feed) diets. Females from the reference group were fed a commercial diet. Females from reference, (+)Lys, and (−)Lys groups were stripped and their eggs divided into 0.4–1.2‐g portions and mixed with sperm (21.4±4.3 μL) from either reference, (+)Lys, or (−)Lys males. The mean weight of Yellow Perch females and mean total weight of ovulated eggs were the greatest in the reference group compared with both (+)Lys and (−)Lys groups. There were no differences in the ratio of weight of eggs to female body weight as well as egg size among groups. There was no difference among treatments in the concentration of free amino acids except glutamic and aspartic acids in Yellow Perch eggs. There was significant effect of female dietary treatments on the egg fertilization rate averaged across all males. The higher fertilization rate was observed in the reference and (+)Lys groups compared with the (−)Lys group. The effect of female dietary treatment on the egg survival was also dependent on the dietary treatment of males.

The Influence of Dietary Lysine on Yellow Perch Maturation and the Quality of Sperm

AbstractThe objective of the present study was to determine whether lysine (Lys) supplementation influences the maturation and sperm quality of Yellow Perch Perca flavescens. Juveniles raised on a formulated commercial diet and weighing approximately 75 g each were randomly distributed into six 400‐L tanks. This experiment included two wheat‐gluten‐based diets in triplicate: (−) Lys (Lys‐deficient) and (+) Lys (Lys‐supplemented; 2.23% in dry feed) diets. In addition, 16 control fish were kept under identical conditions and fed a commercial diet. The weight of males was larger in the control group than in the (+) Lys and (−) Lys groups. The sperm concentration was significantly higher in the control and (+) Lys groups than in the (−) Lys group. Sperm motility was lower in the (−) Lys group than in the control and (+) Lys groups. The control group had significantly higher protein concentration in its seminal plasma than did the (+) Lys and (−) Lys groups. The seminal plasma trypsin inhibitor activity showed the same trend. The seminal plasma free amino acid concentrations of arginine, methionine, threonine, glutamine, alanine, and glycine differed significantly among treatments. This is the first report demonstrating the negative effect of dietary lysine level in plant‐protein‐based diets on reproduction in fish.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

The presence of thiolated compounds allows the immobilization of enzymes on glyoxyl agarose at mild pH values: New strategies of stabilization by multipoint covalent attachment

Abstract Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the ɛ-amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the ɛ-amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilized-stabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4–5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000–50,000-fold more stable than the soluble enzyme).

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low p Kb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.

Sites of Modification of Hemospan, a Poly(ethylene glycol)-Modified Human Hemoglobin for Use As an Oxygen Therapeutic

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.

Structures of potent selective peptide mimetics bound to carboxypeptidase B

This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2 nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1' pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate.

Factors affecting the use of13Cα chemical shifts to determine, refine, and validate protein structures

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13C(alpha) chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13C(alpha) chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13C(alpha) chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13C(alpha) chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2(xi) possible ionization states of the whole molecule, with xi being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshielding of the 13C(alpha) nucleus, indicated that: (i) there is a significant difference in the computed 13C(alpha) chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13C(alpha) nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13C(alpha) chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13C(alpha) chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13C(alpha) chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13C(alpha) chemical shifts (by up to 3.7 ppm), was found for approximately 68% and approximately 63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm < or = rmsd < or = 2.13 ppm), between computed and observed 13C(alpha) chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13C(alpha) chemical shifts and chi(1) torsional angles (given by the vicinal coupling constants, 3J(N-Cgamma) and 3J(C'-Cgamma), is discussed.

Albumin Clusters: Structurally Defined Protein Tetramer and Oxygen Carrier Including Thirty-Two Iron(II) Porphyrins

Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shell albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau1/2) of the 125I-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O2-binding properties (O2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume of O2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.

A Mass Spectrometric Determination of the Conformation of Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.