色甘酸钠对大鼠心肌梗死后心肌凋亡及凋亡蛋白的影响

目的 研究肥大细胞脱颗粒程度对心肌梗死后的心肌凋亡及心肌纤维化的影响,探讨细胞凋亡机制在心肌梗死后心室重构中的作用.方法 建立雄性SD大鼠心肌梗死模型,分成假手术组(n=6)、模型组(n=8)、色甘酸钠组(n=7).术后4周,留取心肌标本行TUNEL染色观察心肌细胞凋亡指数(AI),Masson染色检测心肌胶原容积分数(CVF),Western blot法检测Caspase-3、Bcl-2和Bax蛋白表达.结果 与假手术组相比,模型组、色甘酸钠组在AI、CVF、Caspase-3及Bax蛋白表达量等方面均显著升高(P＜0.05),Bcl-2蛋白表达明显下降;色甘酸钠组与模型组相比,AI、CVF、Caspase-3及Bax蛋白表达量均有所下降(P＜0.05),Bcl-2蛋白有所升高,但两者差异无统计学意义(P=0.127).AI与CVF呈正相关(r=0.769,P＜0.01).结论 心肌梗死后心肌凋亡显著增加,抑制肥大细胞脱颗粒可明显减少心肌凋亡,减轻左心室心肌纤维化程度.     

刺五加对脑出血大鼠细胞凋亡的影响

目的 观察刺五加(AS)对脑出血(ICH)大鼠脑细胞凋亡及其对凋亡调控蛋白Bcl-2、Bax的影响及对ICH脑组织的保护作用.方法 雄性Wistar大鼠随机分为正常组、假手术组、模型组与治疗组.治疗组又分为AS大剂量组和小剂量组.用胶原酶注入大鼠尾状核建立ICH模型.治疗组给予AS干预,在不同时间点处死大鼠,测定血肿周围组织细胞凋亡、Bcl-2和Bax水平.结果 AS治疗组分别于12 h、1、3、7 d凋亡细胞低于模型组(P<0.01,P<0.05).AS治疗组Bcl-2水平分别于12 h、1、3、7 d表达高于模型组(P＜0.01,P＜0.05).Bax于12 h、1、3、7 d时AS治疗组表达低于模型组(P＜0.01,P＜0.05).结论 AS能明显减少ICH后神经细胞凋亡,升高Bcl-2表达水平,降低Bax表达,保护ICH后神经细胞.     

有氧运动对衰老模型大鼠心肌细胞凋亡及Bcl-2和Bax蛋白表达的影响

目的 探讨有氧运动对衰老模型大鼠心肌细胞凋亡及Bcl-2和Bax蛋白表达的影响.方法 建立衰老大鼠模型,随机分为对照组和两组运动组,运动组分别给予6次/w、 3次(隔日)/w游泳训练,每次持续90 min,训练12 w,于末次训练48 h后处死动物,TUNEL法检测心肌细胞凋亡,免疫印迹法检测心肌Bcl-2、Bax的表达,并计算Bax/Bcl-2的比值.结果 与对照组相比,两组运动组心肌细胞凋亡指数(AI)均显著降低(P＜0.01),Bcl-2蛋白表达显著升高(P＜0.01),Bax蛋白表达降低(P＜0.05),Bcl-2/Bax显著升高(P＜0.01);与6次/w运动组相比较,3次/w运动组心肌AI降低(P＜0.05),Bcl-2升高(P＜0.05),Bax降低(P＜0.05),Bcl-2/Bax升高(P＜0.05).结论 长期有氧运动使 Bcl-2/Bax比值优化,降低心肌细胞AI,促进心肌细胞存活,延缓心肌衰老;隔日有氧运动效果更佳.     

益肾化浊方对阿尔茨海默病大鼠Bcl-2/Bax及α分泌酶蛋白可溶性淀粉酶前体蛋白α表达的影响

目的探讨益肾化浊方对阿尔茨海默病(AD)模型大鼠可溶性淀粉酶前体蛋白α(s APPα)、α分泌酶蛋白以及Bcl-2/Bax比值的影响。方法将30只雄性SD大鼠随机分为假手术组、模型组、益肾化浊方组,每组10只。益肾化浊方组灌以中药,假手术组与模型组大鼠灌胃等体积生理盐水,1次/d,共4 w。治疗后取海马标本并采用Western印迹检测大鼠海马中s APPα、α分泌酶以及Bcl-2/Bax比值的表达。结果与假手术组比较,模型组s APPα、α分泌酶蛋白、Bcl-2/Bax比值表达明显降低(P0.05);与模型组相比,益肾化浊方组s APPα、α分泌酶蛋白、Bcl-2/Bax比值明显升高(P0.05)。结论益肾化浊方可通过促进Bcl-2表达、抑制Bax表达提高Bcl-2/Bax比值,上调AD模型大鼠海马中s APPα、α分泌酶蛋白,发挥抑制神经细胞凋亡、抑制Aβ的神经毒性作用。     

芪参益气汤对阿霉素致心力衰竭大鼠心功能及miR133a表达的影响

目的探讨芪参益气汤对阿霉素致心力衰竭(CHF)大鼠心功能及微小核糖核酸133a(miR133a)表达的影响。方法选取清洁级健康雄性Wistar大鼠90只,随机分为对照组、模型组、西药组及中药低、中、高剂量组,各15只。模型组、西药组及中药低、中、高剂量组采用阿霉素尾静脉注射建立CHF模型,对照组大鼠以等量0.9%氯化钠溶液尾静脉注射。模型建立后,模型组和对照组分别予以0.9%氯化钠溶液10mL/kg灌胃给药,每日1次;西药组予以0.25mg/mL卡托普利溶液10mL/kg灌胃给药每日1次;中药低、中、高剂量组分别予以0.8g/mL、1.6g/mL、3.2g/mL芪参益气汤10mL/kg,灌胃给药,每日1次。各组大鼠均给药28d。检测比较各组心脏功能、心肌细胞凋亡指数(AI)、心肌组织B淋巴细胞瘤-2(Bcl-2)蛋白、含半胱氨酸的天冬氨酸蛋白水解酶-9(Caspase-9)表达水平及miR133a、Bcl-2mRNA、Caspase-9mRNA表达水平。结果与模型组比较,其他各组大鼠左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)较低,心肌AI较低,心肌组织Caspase-9蛋白及Caspase-9mRNA表达水平较低,且除LVEDD指标中药中剂量组与中药低剂量组比较差异无统计学意义之外,其余指标均中药低剂量组>中药中剂量组>中药高剂量组和西药组,差异均有统计学意义(P<0.05)。与模型组比较,其他各组大鼠射血分数(EF)、短轴缩短分数(FS)较高,心肌组织Bcl-2蛋白及miR133a、Bcl-2mRNA表达水平较高,且中药低剂量组<中药中剂量组<中药高剂量组和西药组,差异有统计学意义(P<0.05)。结论芪参益气汤能明显改善CHF大鼠的心功能,而且剂量越高效果越好,其作用可能与促进miR133a表达,调节miR133a下游Bcl-2、Caspase-9蛋白表达水平,抑制心肌细胞凋亡有关。     

旋覆花素对阿尔茨海默病模型大鼠脑海马组织Bax,Bcl-2表达的影响

目的 观察旋覆花素对Aβ25 ～35海马内注射所致阿尔茨海默病(AD)模型大鼠海马Bax,Bcl-2表达的影响.方法 30只SD大鼠随机分为对照组、模型组及旋覆花素给药组,Aβ25 ～35海马内注射制备AD大鼠模型,给药组给予旋覆花素26mg·kg-1 ·d-1、模型组给予等量溶剂,分两次灌胃,共21 d.免疫组化及Western印迹测定大鼠海马Bax,Bcl-2表达.结果 模型组海马Bax表达升高,给药组Bax表达下降(P＜0.05).对照组大鼠脑海马组织Bc1-2表达量与模型组及旋覆花素给药组大鼠脑海马组织表达量无明显差别(P＞0.05).结论 旋覆花素主要通过抑制促凋亡基因Bax的表达,从而抑制Aβ诱导的AD模型大鼠脑海马的神经细胞凋亡,而对凋亡抑制基因Bcl-2表达影响不大.     

吡格列酮对高脂血症大鼠主动脉血凝素样氧化型低密度脂蛋白受体1和凋亡蛋白表达的影响

目的 观察吡格列酮对高脂血症大鼠主动脉血凝素样氧化型低密度脂蛋白受体1 (LOX-1)和凋亡蛋白Bax、Bcl-2表达的影响,并探讨其作用机制及LOX-1在吡格列酮调节凋亡蛋白Bax、Bcl-2表达中的作用.方法 清洁型SD大鼠26只,随机分为对照组(n=9)、高脂饮食组(n=17),高脂饮食组喂养12周后再随机分为模型组(n=8)和吡格列酮组(n=9),分别干预4周后,检测各组血脂水平,HE染色观察主动脉病理形态学改变,免疫组织化学法检测主动脉LOX-1、Bax和Bcl-2的表达.结果 高脂饮食组喂养12周后,血脂明显升高(P＜0.01);给药4周后,与模型组比较,吡格列酮组甘油三酯、总胆固醇水平明显降低(P＜0.01),且主动脉血管内皮基本完好,偶有脱落,平滑肌细胞增殖不明显.与对照组相比,模型组主动脉LOX-1和Bax蛋白表达明显升高(P＜0.01),Bcl-2蛋白表达和Bcl-2/Bax比值显著降低(P＜0.01);与模型组相比,吡格列酮组主动脉LOX-1和Bax蛋白表达明显降低(P＜0.01),Bcl-2蛋白表达和Bcl-2/Bax比值明显升高(P＜0.01).结论 高脂饮食可引起主动脉LOX-1和凋亡蛋白Bax、Bcl-2及Bcl-2/Bax比值的改变,而吡格列酮可降低血脂,调节LOX-1和凋亡蛋白表达,抑制内皮细胞凋亡,从而改善内皮细胞功能和动脉粥样硬化病理进程.     

原花青素对缺血再灌注大鼠心肌细胞凋亡及凋亡相关基因蛋白表达的影响

目的:观察原花青素对缺血再灌注大鼠心肌细胞凋亡相关基因半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B型白细胞/2型淋巴细胞样蛋白(Bcl-2)和凋亡相关基因Bcl-2相关X蛋白(Bax)蛋白表达的影响,探讨原花青素在大鼠缺血再灌注心肌细胞凋亡中发挥的作用及可能的调控机制。方法:40只雄性SD大鼠随机等分为4组,即正常组,模型组,原花青素低剂量组[原花青素50 mg/(kg·d)],原花青素高剂量组[原花青素100 mg/(kg·d)],灌胃给药,每天1次,连续2周。末次给药后结扎冠状动脉左前降支(LAD)30 min再灌注120 min,建立大鼠心肌缺血再灌注模型。测定肌酸激酶同工酶(CK-MB)活性、心肌梗死面积;用Western blot检测各组细胞凋亡相关基因Caspase-3、Bcl-2、Bax蛋白表达;原位末端转移酶标记(TUNEL)法检测心肌细胞凋亡。结果:与正常组比较,模型组大鼠CK-MB活性显著增强、心肌梗死面积增大,心肌细胞凋亡指数升高,Caspase-3、Bax蛋白表达增强,Bcl-2蛋白表达、Bcl-2/Bax降低(P0.05);与模型组比较,原花青素高、低剂量组大鼠CK-MB活性显著减弱、心肌梗死面积减小,细胞凋亡指数显著降低,Caspase-3、Bax蛋白表达显著减弱,Bcl-2蛋白表达、Bcl-2/Bax增加(P0.05)。结论:原花青素可拮抗缺血再灌注大鼠心肌细胞凋亡,其机制可能与Caspase-3、Bax蛋白表达降低,Bcl-2表达升高,Bcl-2/Bax比例增加有关。     

肾气丸对非酒精性脂肪性肝炎大鼠Bcl-2/Bax、Fas/FasL信号通路的影响

目的:观察Bcl-2/Bax、Fas/Fas L介导的信号转导通路在高脂饮食诱导的非酒精性脂肪性肝炎大鼠的作用及肾气丸对其的影响。方法:采用高脂饲料连续喂养12周建立大鼠非酒精性脂肪性肝炎模型,同时以肾气丸进行干预12周。HE染色观察大鼠肝组织病理组织学变化,免疫组织化学检测肝组织中Fas、Fas L、Bcl-2、Bax、Caspase-8蛋白的表达;Realtime-PCR法检测肝组织Caspase-8 mRNA表达。结果:模型组大鼠肝组织Bcl-2、Bax、Fas、Fas L蛋白以及Caspase-8 mRNA和蛋白表达均较正常组显著增加(P<0.01),Bcl-2/Bax比值较正常组明显的降低(P<0.05);大鼠肝组织Caspase-8蛋白表达与Fas、Fas L蛋白表达均呈明显正相关(r分别为0.907、0.804,P均<0.01);予以肾气丸干预后,Bax、Fas、Fas L蛋白的表达以及Caspase-8 mRNA和蛋白的表达均较模型组明显的降低(P<0.05),Bcl-2/Bax比值较模型组明显的增加(P<0.05),Bcl-2蛋白的表达与模型组比较差异无显著性意义(P>0.05)。结论:肾气丸可能通过影响Bcl-2/Bax、Fas/Fas L信号转导通路,下调Bax、Fas、Fas L、Caspase-8表达,减少肝细胞的脂性凋亡,从而防治大鼠非酒精性脂肪性肝炎的发生发展。     

Caspase-9、Bcl-2和Bax在基底细胞样乳腺癌组织中的表达及意义

目的 探讨半胱天冬氨酸蛋白酶-9（Caspase-9）、B淋巴细胞瘤/白血病-2（Bcl-2）基因、Bcl-2同源拮抗蛋白/致死蛋白（Bax）基因在基底细胞样乳腺癌（BLBC）发生、发展中的作用。方法 采用免疫组化法检测43例BLBC标本（BLBC组）、57例非BLBC乳腺癌标本（非BLBC组）及60例正常乳腺组织标本（正常组）Caspase-9、Bcl-2及Bax蛋白的表达水平,分析三者表达的相关性及其与BLBC临床病理特征的关系。结果 BLBC组、非BLBC组Bcl-2表达率均明显高于正常组,Caspase-9和Bax表达率均低于正常组,P均〈0.05;Caspase-9、Bcl-2及Bax表达与BLBC淋巴结转移及临床分期相关,与年龄、肿瘤大小无关,Caspase-9与Bax表达呈正相关（r=0.572,P〈0.05）,Caspase-9与Bcl-2、Bax与Bcl-2表达均呈负相关（r=-0.381,r=-0.408,P〈0.05）。结论 Caspase-9与Bax在BLBC组织中均呈低表达,Bcl-2呈高表达;三者在BLBC的发生、发展中共同发挥作用。     

In vivo protein stabilization based on fragment complementation and a split GFP system

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.     

Multimolecule test-tube simulations of protein unfolding and aggregation

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.     

蛋白质棕榈酰化修饰与心血管疾病

蛋白质棕榈酰化修饰是一种广泛存在于生物体内的蛋白质翻译后脂质修饰。在大多数情况下,棕榈酸主要通过不稳定的硫酯键共价结合到蛋白质底物特定的半胱氨酸残基上,从而发生S-棕榈酰化修饰。由于硫酯键在一定的条件下会水解,引起蛋白质底物发生去棕榈酰化修饰,因此S-棕榈酰化修饰通常是可逆的。S-棕榈酰化修饰也与许多心血管疾病的发生和发展密切相关。本文综述了蛋白质棕榈酰化修饰和去棕榈酰化修饰过程,以及蛋白质棕榈酰化修饰与心律失常、动脉粥样硬化、肺动脉高压、心力衰竭和血栓性疾病等心血管疾病之间的关系,为心血管疾病的治疗提供新的方向。     

uPA、P-选择素蛋白在乳腺癌中的表达及意义

目的 探讨uPA与P-选择素蛋白在乳腺癌浸润、转移中的作用以及二者的关系。方法　采用免疫组化SP法检测uPA与P-选择素蛋白在乳腺纤维腺瘤、乳腺导管内癌及浸润性导管癌组织中的表达。结果 随着乳腺组织的恶性转化、浸润及淋巴结转移,uPA、P-选择素蛋白均呈高表达趋势(P<0.05),二者之间有明显的相关性(r=0.547,P<0.01)。结论 uPA与P-选择素蛋白参与了乳腺癌的浸润及淋巴结转移,联合检测二者,对乳腺癌的预后判断有一定的参考价值。     

Characterization of prolactin- and growth hormone-binding proteins in milk and their diversity among species

The present study was undertaken to identify and characterize the diversity and species distribution of soluble prolactin binding-protein (PRL-BP) and growth hormone-binding protein (PRL-BP) in mammalian milk. We previously divided mammalian serum GH-BP into four main groups and identified a GH-BP with shared lactogenic/somatogenic properties in rabbit, horse, dog, pig and cat (Type III species). Here we describe PRL-BP in milk of Type III species and show it is relatively conserved within the group, having similar characteristics in terms of binding affinity for hGH (0.74−5.5×10¹⁰ M⁻¹), specificity towards the lactogenic hormones and molecular weight (35 kDa), except for the more heterogeneous pig milk (43 to 88 kDa) Furthermore, high affinity PRL-BP was also demonstrated in sheep milk, having pure lactogenic specificity and an M_r of 35 kDa. Human milk contained a high affinity PRL-BP/GH-BP, which was recognized by both hPRL and hGH and also having an M_r of 35 kDa. In rabbit milk a separate GH-BP was also detected; it was clearly distinguished from the corresponding milk PRL-BP on the basis of its M_r of 44 kDa (vs. 32 kDa for PRL-BP), its shared lactogenic/somatogenic hormonal specificity (vs. purely lactogenic for PRL-BP) and also on the basis of its relative resistance to heating at 56°C for up to 3 h, while PRL-BP activity was completely destroyed within 30 min. This diversity of milk PRL-BP and GH-BP among mammalian species fits in with our earlier classification of serum GH-BP and also with the reported evolutionary rates of PRL and GH; this suggests these BPs may play important species-specific roles in the suckling newborn and/or maternal mammary gland, in keeping with the functions described for GH-BP.     

Molecular in situ topology of Aczonin/Piccolo and associated proteins at the mammalian neurotransmitter release site

The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.     

Folding pathways of proteins with increasing degree of sequence identities but different structure and function

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G_A (a 3-α helix fold) and G_B (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G_A domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G_Bs, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.     

From the Cover: In-cell thermodynamics and a new role for protein surfaces

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.Classic theories about the effects of complex environments consider only hard-core repulsions (volume exclusion) and so predict entropy-driven protein stabilization (1–3). Here, we use the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as a model to test this idea in living cells. SH3 exists in a dynamic equilibrium between the folded state and the unfolded ensemble (4). This two-state behavior (5) is ideal for NMR-based studies of folding. Fluorine labeling (6) of its sole tryptophan leads to only two ¹⁹F resonances (7): one from the folded state, the other from the unfolded ensemble (Fig. 1A). The area under each resonance is proportional to its population, ρ_f and ρ_u, respectively. These populations are used to quantify protein stability via the modified standard state free energy of unfolding,ΔGU,T°′=−RTlnρUρF,[1]where R is the gas constant and T is the absolute temperature. Furthermore, the width at half height of each resonance is proportional to the transverse relaxation rate, which is an approximate measure of intermolecular interactions (8–10). Thus, this simple system yields both quantitative thermodynamic knowledge and information about interactions involving the folded state and the unfolded ensemble.Open in a separate windowFig. 1.Fluorine spectra acquired at 298 K, in buffer (A) and cells (B). The blue trace is from the postexperiment supernatant and shows that the red spectrum arises from protein inside cells. Stability curves (C) in buffer (black), in cells (red and green), and in 100 g/L urea (magenta). In-cell metabolite correction and analysis of uncertainties are discussed in Results and Discussion and Materials and Methods, respectively. Shaded regions are 95% confidence intervals. Error bars for buffer are smaller than the labels and represent the SD of three trials. Error bars for the in-cell data at 273, 298, and 313 K represent the SD of three trials. Stability in buffer (black) and solutions of 100 g/L BSA (blue) and lysozyme (red) at different pH values (D–F). The curve for buffer from C is reproduced in D. The net charges on SH3, BSA, and lysozyme (based on sequence) are shown. Error bars (298 K) represent the SD from three trials. Appearance of new resonances in the pH 3 BSA sample prevented extraction of thermodynamic parameters.To assess the enthalpic (ΔHU°′) and entropic (ΔSU°′) components, we measured the temperature dependence of ΔGU°′. These data were fitted to the integrated Gibbs–Helmholtz equation (11), assuming a constant heat capacity of unfolding, ΔCp,U°:ΔGU,T°′=ΔHU,Tref°′−TΔSU,Tref°′+ΔCp,U°′[T−Tref−T⁡lnTTref],[2]where T_ref is either the melting temperature, T_m (where ρ_f = ρ_u), or the temperature of maximum stability, T_s (where ΔSU°′ = 0) (11).     

Structural relationships among proteins with different global topologies and their implications for function annotation strategies

It has become increasingly apparent that geometric relationships often exist between regions of two proteins that have quite different global topologies or folds. In this article, we examine whether such relationships can be used to infer a functional connection between the two proteins in question. We find, by considering a number of examples involving metal and cation binding, sugar binding, and aromatic group binding, that geometrically similar protein fragments can share related functions, even if they have been classified as belonging to different folds and topologies. Thus, the use of classifications inevitably limits the number of functional inferences that can be obtained from the comparative analysis of protein structures. In contrast, the development of interactive computational tools that recognize the “continuous” nature of protein structure/function space, by increasing the number of potentially meaningful relationships that are considered, may offer a dramatic enhancement in the ability to extract information from protein structure databases. We introduce the MarkUs server, that embodies this strategy and that is designed for a user interested in developing and validating specific functional hypotheses.