去甲肾上腺素预处理对大鼠供心热休克蛋白70、一氧化氮及一氧化氮合酶表达的影响

目的:探讨去甲肾上腺素预处理是否可诱导心肌热休克蛋白70(HSP70)的合成,并研究其对供心一氧化氮(NO)、一氧化氮合酶(NOS)的影响,探讨去甲肾上腺素预处理心肌保护作用机制。方法:Wistar大鼠18只,分为2组:对照组(C,n=9),腹腔注射0.9%氧化钠注射液0.5 mL,24 h后取离体心脏灌注(Histidine-tryptophan-ketoglutarte,HTK)心脏保护液,4℃保存3 h后建立Langendorff离体心脏灌注模型,灌注(Krebs-Henseleit,K-H)液2 h;实验组(E,n=9)腹腔注射重酒石酸去甲肾上腺素(溶于0.9%氯化钠液中)3.1μmol/kg(0.53 mg/kg),腹腔注射24 h后取离体心脏,处理方法同C组。测定心肌HSP70、NO、NOS的含量以及相关生化指标并做统计学处理比较。结果:HSP70含量E组较C组明显增高(P<0.01),NO、NOS的含量E组较C组明显增多(P<0.01),生化指标E组明显优于C组。结论:去甲肾上腺素预处理能诱导供心心肌组织HSP70、NO、NOS高表达,其对供心具有明显的保护效应,并且其促进心肌NO、NOS的表达,这可能是去甲肾上腺素预处理发挥供心保护作用的机制之一。     

黄芪对病毒性心肌炎小鼠热休克蛋白70及Bcl-2、Bax基因表达的影响

目的:研究黄芪对病毒性心肌炎(VMC)小鼠热休克蛋白(HSP)70及凋亡相关基因蛋白产物表达的影响。方法:体外培养乳鼠心肌细胞,用Western Blot观察心肌细胞HSP70、Bcl-2及Bax变化;120只Balb/c小鼠随机分为正常对照组、病毒组以及黄芪干预组,采用腹腔接种柯萨奇病毒制做VMC小鼠模型,免疫组化检测各组小鼠Bcl-2和Bax基因表达的情况,并用图像分析系统进行分析。结果:黄芪干预组心肌细胞HSP70及Bcl-2表达明显高于病毒组,2组Bax表达无差异;黄芪干预组心肌组织Bcl-2表达明显高于病毒组,2组Bax表达亦无差异。结论:黄芪可能通过上调心肌细胞Bcl-2、HSP70的表达来抑制病毒所致的心肌细胞凋亡。     

热休克蛋白70对未成熟心肌间质保护作用的研究

目的探讨热休克蛋白70(HSP70)对未成熟心肌间质的影响。方法健康新生长耳大白兔随机分为2组。对照组:腹腔注射生理盐水0.4ml,注射后24h取离体心脏,常规建立Langendorff离体心脏灌注模型,灌注15min转为工作心15min后停灌45min,恢复灌注15min改为工作心30min;观察组:腹腔注射重酒石酸去甲肾上腺素,24h后取离体心脏,方法同对照组。测定心脏舒张功能指标、心肌细胞中HSP70含量、心肌羟脯氨酸(HP)和血清内皮素(ET)含量。结果HSP70含量观察组明显高于对照组;观察组HP含量高于对照组(P<0.05),ET含量低于对照组(P<0.05)。结论HSP70可明显减轻未成熟心肌间质缺血再灌注损伤。     

金属硫蛋白对离体心脏心肌间质的保护作用

目的 探讨金属硫蛋白(MT)对离体心脏心肌间质的影响。方法 Wistar大鼠16只,分为2组:对照组(C,n=8),腹腔注射蒸馏水0.5ml 24h后取离体心脏行离体灌注(Langendorff模型),测定心功能,然后灌注HTK心脏保护液,4C保存3h后再行Langendorff灌注25min;实验组(E,n=8)腹腔注射3.6％硫酸锌(1.5 ml/kg)24 h后取离体心脏,处理方法同C组。以心肌细胞中MT含量、血流动力学指标、心肌组织羟脯氨酸(HP)含量、内皮素(ET)含量和心肌超微结构等作为观察指标。结果MT含量E组与C组比较明显增高;E组心功能恢复方面优于C组(P<0.05),HP含量优于C组(P<0.01),ET含量低于C组(P<0.01),心肌超微结构损伤较C组明显减轻。结论MT对供心心肌间质具有保护作用。     

猪心肌缺血后处理对心肌细胞凋亡及Bcl-2、BaX蛋白表达的影响

目的探讨猪心肌缺血后处理对细胞凋亡及Bcl-2、Bax蛋白表达的影响。方法24只小型约克猪被随机分为4组,建立体外循环全心缺血再灌注模型。A组未行心肌缺血处理,B组于升主动脉阻断前进行心肌缺血预处理,C组再灌注开始前行缺血后处理,D组升主动脉阻断前行心肌缺血预处理及再灌注开始前行心脏缺血后处理,观察各组心肌细胞凋亡率及Bcl-2、Bax蛋白表达。结果心肌细胞凋亡率B组(10.46±0.91)%、C组(9.68±0.59)%和D组(11.35±1.37)%显著低于A组(19.75±1.81)%,差异有统计学意义(P<0.05);C组与B组或D组比较无统计学意义(P>0.05)。Bcl-2蛋白表达B组、C组和D组明显高于A组(P<0.05),Bax蛋白表达B组、C组和D组)明显低于A组(P<0.05)。B组、C组和D组间Bcl-2、Bax蛋白表达无统计学意义(P>0.05)。心肌细胞凋亡率与Bax/Bcl-2比值呈正相关(r=0.926,P<0.01)。结论缺血后处理可上调Bcl-2蛋白表达,下调Bax蛋白的表达,抑制心肌细胞凋亡,减轻缺血再灌注损伤。     

运动预适应对急性心肌缺血大鼠心肌细胞凋亡及凋亡蛋白表达的影响

目的探讨运动预适应(EP)对非动脉硬化大鼠及动脉硬化大鼠急性心肌缺血心肌细胞B细胞淋巴瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)蛋白表达及半胱氨酸蛋白酶(Caspase)-3蛋白的影响。方法选取7周龄SD大鼠90只,随机分为5组:对照组(A组)、急性心肌缺血组(B组)、动脉硬化+急性心肌缺血组(C组)、EP+急性心肌缺血组(D组)、动脉硬化+EP+急性心肌缺血组(E组),其中A组10只,其余各组20只。C、E组予维生素D3连续灌胃4 d,喂高脂饲料(猪油、胆固醇、胆酸、丙基硫氧嘧啶)6 w制备动脉硬化模型。B、C、D、E组腹腔注射3 mg/kg异丙肾上腺素(ISO),复制大鼠急性心肌缺血损伤模型。D、E组建立EP大鼠模型进行间歇性游泳运动,以尾部负重体重的3%重物。建模后24 h内采用脊髓脱臼法处死大鼠,取左心室心肌组织,采用免疫组化检测Bcl-2及Bax蛋白的表达,Western印迹检测Caspase-3蛋白。结果与A组比较,B、C、D、E组心肌细胞Bcl-2的表达均显著降低(P<0.05),而Bax表达均显著升高(P<0.05);B组与C组心肌细胞Bcl-2的表达差异无统计学意义(P>0.05),但C组Bax的表达显著升高(P<0.05);D组、E组心肌细胞Bcl-2的表达较B组、C组显著升高(P<0.05),而Bax的表达显著降低(P<0.05);与D组比较,E组Bcl-2的表达显著降低,而Bax的表达显著升高(P<0.05)。与A组相比,B、C、D、E组心脏Caspase-3蛋白表达水平显著升高(P<0.01);与B组、C组相比,D组、E组心脏Caspase-3蛋白表达水平显著降低(P<0.01)。与D组比较,E组心脏Caspase-3蛋白表达水平无明显差异(P>0.05);与A组比较,B、C、D、E组细胞凋亡指数(AI)显著增高(P<0.05),其中C组AI最高;与B组、C组比较,D组、E组AI显著降低(P<0.05),而D组比E组AI稍低(P<0.05)。结论EP可能改善急性心肌缺血或是在合并动脉硬化基础上急性心肌缺血大鼠的心肌细胞凋亡,可能基于凋亡相关基因Bcl-2表达升高、Bax表达降低调控及下调Caspase-3蛋白所致。     

银杏叶提取物对大鼠急性心肌缺血再灌注时心肌细胞凋亡及凋亡相关基因表达的影响

目的 探讨银杏叶提取物(EGB)对大鼠心肌缺血再灌注时心肌细胞凋亡及凋亡相关基因表达的影响.方法 采用结扎左冠状动脉前降支(LAD)30 min,再灌注2 h复制大鼠心肌缺血再灌注模型,分别以缺口末端标记法(TUNEL)及免疫组织化学法检测心肌凋亡细胞、心肌细胞Bcl-2、Bax基因的蛋白表达的变化.结果 缺血再灌注(IR)组心肌细胞凋亡数量显著高于假手术组(P<0.01),EGB组心肌细胞凋亡明显受到抑制(P<0.01).IR组和EGB组Bax、Bcl-2、P53基因蛋白表达均明显增加(P<0.01);EGB明显促进Bcl-2基因表达,同时抑制Bax、P53基因表达(P<0.01),Bcl-2和Bax的比值也随之升高.结论 EGB可明显下调Bax和P53基因的蛋白表达,上调Bcl-2基因的蛋白表达,从而显著抑制心肌缺血再灌注损伤(MIRI)后心肌细胞凋亡.     

丙泊酚对大鼠离体缺血/再灌注心肌细胞凋亡和Bcl-2/Bax蛋白表达的影响

目的研究丙泊酚对大鼠离体缺血/再灌注心肌细胞凋亡的影响及其机制。方法 SD大鼠30只,随机分为丙泊酚组与对照组,制备大鼠离体心肌缺血/再灌注(MI/R)模型,在再灌注开始时分别给以丙泊酚(25μmol/L,以950 mL/L乙醇为溶剂)或950 ml/L乙醇(3μL/h)。再灌注结束后,将缺血区剪下,制备石蜡切片,采用TUNEL技术检测心肌凋亡,免疫组化技术检测心肌组织的Bcl-2和Bax水平。结果丙泊酚组与对照组相比,心肌细胞凋亡明显增多,分别为(3 5.2±6.7)%(、5.2±0.8)%(t=28.1,P<0.01);Bcl-2表达降低,分别为5.7±1.3、1.8±0.8(t=11.9,P<0.01);Bax增高,分别为9.1±1.3、5.9±1.3,(t=11.9,P<0.01)。结论大剂量丙泊酚促进大鼠离体缺血/再灌注心肌细胞凋亡,提高Bax蛋白表达,降低Bcl-2蛋白表达。丙泊酚对Bcl-2/bax的调节可能是其促凋亡的主要机制之一。     

缺血后适应对大鼠移植心脏心肌细胞凋亡的影响

目的:观察缺血后适应对大鼠移植心脏缺血再灌注时心肌细胞凋亡及对抗凋亡基因Bcl-2(简称Bcl-2)、凋亡基因Bax(简称Bax)蛋白表达的影响。方法:30只Lewis大鼠随机分成3组,每组10只,供体、受体各5只。假手术组:仅行腹部开关手术,不行心脏移植;模型组:行心脏移植术;后适应组:行心脏移植手术,心脏复灌前进行缺血后适应,即再灌注10 s、关闭10 s,循环3次。实验结束后取心肌标本行生物素切口末端标记法(TUNEL)检测,并采用蛋白免疫印迹(Western Blot)方法检测Bcl-2、Bax蛋白表达。结果:假手术组无心肌细胞凋亡,后适应组心肌细胞凋亡指数明显低于模型组,差异有统计学意义(P<0.01)。Western Blot显示:模型组Bcl-2、Bax蛋白表达水平与假手术组比升高(P<0.05);后适应组Bcl-2蛋白表达水平与模型组相比明显升高(P<0.01),而Bax蛋白表达水平降低(P<0.05),差异均有统计学意义。结论:缺血后适应对大鼠移植心脏缺血再灌注心肌能够上调Bcl-2蛋白表达、下调Bax蛋白表达,并可减少缺血再灌注引起的心肌细胞凋亡。     

金属硫蛋白表达对未成熟心肌间质保护作用的研究

目的 :探讨诱导金属硫蛋白 (MT)在未成熟心肌中的表达对缺血 再灌注未成熟心肌间质的影响。方法 :采用Langendorff离体灌注模型 ,分为 4组 :对照组 (C ,n =9) ,腹腔注射蒸馏水 0 3mL ,按注射后时间 12h、2 4h和 4 8h取离体心脏 ,灌注KH液 15min转为工作心 15min ,全心停灌注 4 5min ,恢复灌注 15min改为工作心 30min ;E1 2h组 (n =6 )、E2 4h 组 (n =6 )、E4 8h 组 (n =6 )各组分别按腹腔注射 3 6 %ZnSO4 (1 5mL kg)后 12h、2 4h和 4 8h取离体心脏 ,常规建立Langendorff灌注模型 ,方法同C组。以心肌细胞中MT含量、血流动力学指标、心肌组织羟脯氨酸 (HP)含量、内皮素 (ET)含量作为观察指标。结果 :腹腔注射ZnSO4 后 12hMT开始表达 ,2 4h达高峰 ,4 8h仍在高表达水平。MT含量在E2 4h、E4 8h组与C、E1 2h组比较明显增高 ;E2 4h、E4 8h组在心功能恢复方面均优于C组和E1 2h组 (P <0 0 5 ) ,在HP含量优于C组和E1 2h组 (P <0 0 1) ,在ET含量低于C组和E1 2h组 (P <0 0 1)。结论 :腹腔注射ZnSO4 可诱导心肌MT长时间表达 ,MT可减轻未成熟心肌间质的缺血 再灌注损伤。     

A thermodynamic definition of protein domains

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.     

Treatment of homozygous protein C deficiency with subcutaneous protein C concentrate

Subcutaneous protein C concentrate (Immuno, Vienna) was used to treat a child with homozygous protein C deficiency who was formerly treated with intravenous protein C concentrate. After 3000 units subcutaneous protein C concentrate (250 iu/kg), protective protein C levels were maintained for 48h after infusion, with peak levels at 12h. Subcutaneous protein C concentrate is given every third day and is well tolerated by the patient. No thrombotic events have occurred. We conclude that subcutaneous administration of protein C concentrate is a valuable therapeutic option in the long-term management of homozygous protein C deficiency and avoids the potential hazards of long-term central venous lines.     

Maps of protein structure space reveal a fundamental relationship between protein structure and function

To study the protein structure–function relationship, we propose a method to efficiently create three-dimensional maps of structure space using a very large dataset of > 30,000 Structural Classification of Proteins (SCOP) domains. In our maps, each domain is represented by a point, and the distance between any two points approximates the structural distance between their corresponding domains. We use these maps to study the spatial distributions of properties of proteins, and in particular those of local vicinities in structure space such as structural density and functional diversity. These maps provide a unique broad view of protein space and thus reveal previously undescribed fundamental properties thereof. At the same time, the maps are consistent with previous knowledge (e.g., domains cluster by their SCOP class) and organize in a unified, coherent representation previous observation concerning specific protein folds. To investigate the function–structure relationship, we measure the functional diversity (using the Gene Ontology controlled vocabulary) in local structural vicinities. Our most striking finding is that functional diversity varies considerably across structure space: The space has a highly diverse region, and diversity abates when moving away from it. Interestingly, the domains in this region are mostly alpha/beta structures, which are known to be the most ancient proteins. We believe that our unique perspective of structure space will open previously undescribed ways of studying proteins, their evolution, and the relationship between their structure and function.     

Simplified protein design biased for prebiotic amino acids yields a foldable,halophilic protein

A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment.     

超敏C-反应蛋白和C-反应蛋白的测定对SARS的诊断价值

目的　研究血清超敏C -反应蛋白 (hs -CRP )和C -反应蛋白 (CRP)对严重急性呼吸综合征 (SARS)的诊断价值。方法　SARS病人 2 0例、细菌性肺炎病人 2 0例、健康对照 2 0例 ,血清hs -CRP和CRP采用胶乳免疫比浊法全自动定量测定。结果　hs -CRP和CRP测定结果分别为 :健康对照组 (0 6 9± 0 6 2 )mg/L和 (4 4± 0 9)mg/L、细菌性肺炎组 (10 79± 1 36 )mg/L和 (98 0± 2 8 9)mg/L、SARS组 (3 16± 3 72 )mg/L和 (11 0± 9 6 )mg/L。三组间差异均有显著意义 (P <0 0 1)。结论　SARS病人和细菌性肺炎病人早期血清hs -CRP和CRP均升高 ,但细菌性肺炎病人升高更加显著 ,比SARS组分别增加 2 4倍和 7 9倍 ,对SARS与细菌性性肺炎的鉴别诊断有重要意义     

Associations of protein C and protein S with serum lipid concentrations

Difficulties in the laboratory measurement of protein C and protein S levels cause problems in the diagnosis of deficiency states in individual patients and may complicate estimation of the prevalence of these states in the general population. Some difficulties may be due to unappreciated influences affecting the measured levels of proteins C and S. We measured protein C activity and antigen, total and free protein S antigen, and serum total cholesterol, high-density cholesterol and triglyceride in a community-based study of 150 adults (73 male, 77 female), age range 23–80 years. Participants were identified from the list of a single general practice by stratified random sampling within sex and decade of age. Protein C activity and antigen were strongly associated with serum lipids, mean levels increasing by approximately 0.25 u/ml as total cholesterol and triglyceride concentration each rose from the 5th to 95th centile. Total protein S antigen concentration was associated with total cholesterol, the mean rising by over 0.1 u/ml as total cholesterol increased from the 5th to the 95th centile, whilst a similar rise in triglyceride was associated with an increase in mean free protein S of more than 0.3 u/ml. Overall, physiological variation in total cholesterol and triglyceride concentration was associated with significant variation in protein C and protein S levels, independent of age and sex, suggesting that it is important to take serum lipids into account when investigating patients for protein C or protein S deficiency. Failure to do so may be misleading in some circumstances.     

De novo design of protein homodimers containing tunable symmetric protein pockets

Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.

Cyclic two-fold (C2) symmetric molecules are common in biology and medicine, such as HIV protease inhibitors (1), iron sulfur clusters (2), and the chlorophyll special pair found in photosynthetic reaction centers (3). To bind such compounds, C2 symmetric protein homodimers are advantageous because each protein monomer can make identical interactions with an asymmetric unit of the small molecule. There are many C2 symmetric protein structures in nature, but it is not straightforward to re-engineer them to bind arbitrary C2 symmetric small molecules unless they happen to contain an interior cavity with the correct size and shape. 4-Helix bundles have been engineered to bind chlorophyll dimers (4), di-nuclear metals (5), and iron-sulfur clusters (6), and binding of a C3 symmetric molecule has been achieved using C3 symmetric helical bundles (7). However, the size and shape of binding pockets available in the interior of helical bundles is limited, as interactions between the helices are also important for stability, and hence they cannot be too far apart. A large set of protein scaffolds with C2 symmetric binding pockets spanning a wide range of sizes and shapes that can be functionalized without compromising stability could enable the creation of new enzymes, therapeutics, and light harvesting proteins, but neither such sets nor methods to generate them currently exist.We set out to develop a general solution to the challenge of creating scaffold proteins for binding C2 symmetric ligands. Our approach builds on recent work in the field of de novo protein design centered around robust alpha-helical repeat proteins (8, 9) that have been adapted to create higher order cyclic oligomers (10) and nanocages (11), chemically induced protein switches (12), and proteins binding specific mineral surfaces. In particular, circular tandem repeat proteins, or “toroids,” have been designed in which the curvature of the repeat proteins is chosen to enable closure into ring-like structures with a large circular central cavity (9). We aimed to design repeat proteins that could similarly house a central cavity, but with a wide range of elliptical, rather than perfectly circular, shapes to enable binding of a wider range of C2 symmetric ligands. We chose an overall design architecture consisting of repeat proteins which curve around a central axis that are docked into C2 symmetric homodimers surrounding an elliptical central cavity (Fig. 1A). By employing repeat proteins with minimal rise along the superhelical axis from one repeat unit to the next, we favor C2 arrangements with the ends of the two monomers in contact. Advantages of this conception are that the cavities can be vastly diverse in size, shape, and chemical composition (lined with different sidechain functional groups). Additionally, as the cavity lining residues are on the exterior of the monomers, the protein hydrophobic core is separated from the binding pocket; as such, functionalization to create binding interactions for specific compounds is unlikely to destabilize either the monomers or the dimer interface.Open in a separate windowFig. 1.Design strategy. (A) Schematic of design pipeline from curved repeat protein (Left) to symmetric homodimers (Center) to C2 symmetric ligand binders (Right). Color gradient represents the protein chain direction from N terminus (blue) to C terminus (red). Hypothetical C2 symmetric ligands are shown in gray. (B) Examples of repeat proteins sampling different curvatures. The helical symmetry axis of the proteins is aligned to the z axis, and an xyz axis is depicted to show that we are looking directly down the z axis. (C) A histogram of helical curvature for 1 million backbones made with our biased sampling method (orange) or without (blue). (D) A single monomer (Left) can be docked into various rigid body orientations to create homodimers (Right) with diverse central cavities. This docking approach can create head to tail (light gray box) or head to head + tail to tail (dark gray box) homodimers. (E) Examples of homodimers featuring a range of cavity sizes and shapes. Each dimer is based on a different monomeric curved repeat protein. The top row shows proteins looking down the central cavity, depicted as backbone ribbon representation for both chains and with surface mesh on chain B. The Bottom row shows a side view slicing through the protein to illustrate the shape of the central cavity, depicted as surface representation. Head to tail homodimers are shown in a light gray box and head to head + tail to tail homodimers are shown in a dark gray box. The C2 symmetry axis of the protein homodimer is indicated with a blue axis.To implement this strategy to design C2 symmetric protein homodimers containing central cavities, we first set out to create a diverse library of monomeric units to dock into various symmetric homodimer orientations. Previous efforts to design diverse repeat proteins (8) did not produce many structures with shapes suitable to create the C2 symmetric homodimers with the elliptical central cavities that we envisioned. Because of the lack of existing protein monomers, we began by generating a set of helical repeat protein monomers with structures specifically tailored for building C2 symmetric binding pockets. We selected a range of superhelical curvature, rise, and radius parameters, such that a four-unit repeat protein would approximate a half-circle and the resulting dimer would form an ellipse. Superhelical curvature and rise correspond to the rotation around and translation along the central superhelical axis per repeat unit respectively, and radius is the distance of the protein from this axis; these quantities are calculated from the center of mass of one repeat unit to the center of mass of the next repeat unit (see SI Appendix, Fig. S1 for schematic) using the RepeatParameterFilter within the Rosetta macromolecular modeling suite (13). Model building to approximate a half circle suggested the rotation between each repeat should be between 0.7 rad and 1.1 rad, the rise less than 1.5 Å per repeat, and the radius between 10 Å and 22 Å. We hypothesized that, when docked into dimers, proteins with these parameters would create pockets that could accommodate ligands of diverse sizes and shapes. Fig. 1D and E and SI Appendix, Fig. S2 illustrate how radius, curvature, and rise control the shape of repeat proteins and highlight the type of monomeric proteins we aimed to make.Repeat protein design as previously described (8) relies on Rosetta Monte Carlo fragment assembly approaches to explore repeat protein space (14). Unbiased sampling rarely yields repeat proteins with our desired helical parameters and only in cases where the lengths of the two helices in a repeat differ by 6–7 residues (SI Appendix, Fig. S3). We thus developed methods for biasing fragment assembly toward desired regions of repeat protein parameter space; at each fragment insertion (made identically in each repeat unit), the deviation from a target set of superhelical parameters is computed and the sum of these deviations is added to the coarse-grained score function previously used. With this biased assembly protocol, we were able to focus sampling on repeat protein structures with the desired superhelical parameters at all combinations of helix length (see Fig. 1C and SI Appendix, Figs. S3 and S4); almost all trajectories with the biased fragment assembly protocol generate proteins with superhelical parameters in the desired range (see SI Appendix, Fig. S3). After filtering out poorly packed structures (SI Appendix, Fig. S5), we obtained far more curved repeat proteins at all helix length combinations using the biased fragment assembly protocol (see SI Appendix, Fig. S3; there were a higher fraction of poorly packed structures with the helical parameter bias terms on, and a lower weight on these terms could increase efficiency). We were able to generate 100,000 curved repeat protein backbones to use for subsequent design, a large increase over the handful of curved repeat proteins previously published (8).These backbones were subjected to combinatorial sequence optimization using a RosettaScripts FastDesign protocol with repeat protein symmetry (applied through the RepeatProteinRelaxMover), which makes identical moves to each repeat unit during sequence design and minimization. The designs were then extended or shortened by up to half a repeat unit based on the energy per residue of the terminal helix to eliminate terminal helices that may be disordered due to limited contacts to the rest of the structure. The top 12,000 designs based on a combination of energy, packing, and sequence-structure agreement, were submitted for Ab Initio folding simulations. Designs for which the sequence strongly encoded the structure in de novo structure prediction calculations (see Methods; the lowest energy structures are close to the designed structure), 2,500 in total, were used in subsequent docking and design calculations.We next set out to create C2 symmetric homodimers with central cavities using these 2,500 curved repeat proteins. We extended a previous symmetric docking approach (10) by adding a requirement that the docks create one of two classes of closed circular structure with either two N to C-terminal interfaces (head to tail dimer) or both N to N and C to C-terminal interfaces (head to head + tail to tail dimer). This docking protocol generated 1 million docked structures. We subsequently removed docks that had small interfaces (less than 10 contacting residues) that were likely to form weak interfaces, as well as docks that had excessively large interfaces (greater than 24 contacts) that could lead to poor behavior before dimerization due to having many exposed hydrophobic residues. This yielded a set of about 100,000 docks for both classes of homodimers that were subjected to interface sequence optimization using a RosettaScripts FastDesign protocol (15) with C2 symmetry (16). Fig. 1D shows how a single monomer can be docked into many distinct orientations creating diverse central cavities, and Fig. 1E shows examples of the diversity of proteins and pockets that can be achieved by docking diverse monomers into various C2 symmetric orientations. The top 1,200 (head to tail) and 2,000 (head to tail + tail to tail) homodimer designs were selected based on a combination of interface energy, interface shape complementarity, and buried unsatisfied hydrogen bonds (17) for both classes of closed circular homodimers (see SI Appendix, Fig. S6 for design flowchart).With the ability to generate these proteins computationally, we set out to characterize a diverse set of examples (see SI Appendix, Fig. S7) with pockets varying in volume and shape (see SI Appendix, Fig. S8), approximated through the three principal axes, which were calculated on poly-alanine backbones to represent the maximum possible size of the pockets. In total, we characterized 101 designs including 77 head to tail dimers and 24 head to head + tail to tail dimers. Forty-four of the designs expressed enough soluble, well-behaved, protein for further characterization. Five of these designs were determined to be soluble aggregate by subsequent analysis. Of the 39 remaining proteins, 38 were characterized by circular dichroism (CD) (18), and of these, 36 were found to be helical and hyperstable (maintaining 80% helicity on average at 95 °C). All 36 of these proteins had nearly identical CD spectrums upon cooling back to 25 °C (data for seven designs are shown in Fig. 2). One design characterized by CD appeared helical as expected, but was not hyperstable, while another had low helical signal.Open in a separate windowFig. 2.Biophysical characterization. Left: design names along with the helical parameters (rise, radius, and curvature) of the associated protein monomer. Second column; design models depicted as ribbon backbones colored from blue (N terminus) to red (C terminus). Third column; normalized ultraviolet absorbance (A280) obtained during SEC-MALS, followed by circular dichroism scans from 200 to 260 nm at 25 °C, 95 °C, and 25 °C post heating. Fourth column; predicted SAXS profiles overlaid on experimental SAXS data points for scattering vector (q, from 0 to 0.25) vs. intensity (I).We used small angle X-ray scattering (SAXS) (19, 20) to characterize the 37 designs that appeared helical by CD (including the one with low stability); of these, 31 had experimental SAXS profiles that closely matched profiles predicted for the corresponding design model (19, 21), suggesting that they have the correct ellipsoidal shape in solution (the first six rows of Fig. 2 show representative examples). Taken together, the CD and SAXS data suggest that 31% of designs (31 of 101) are well-expressing soluble dimers with shapes consistent with the design models. See SI Appendix, Table S1 for characterization of all 101 designs.We determined crystal structures for three designs (see Fig. 3). One of these, design D_3_337, is hyperstable and helical by CD, but the SAXS data suggests it dimerizes to a different shape than designed (Fig. 2, bottom row; Table S1). For this design, the monomer rather than the dimer crystalized (Fig. 3C; Protein Data Bank PDB: 7RMY); the Ca rmsd to the design model of the monomer structure is 2.75 Å, demonstrating control over the shape of repeat proteins in the absence of the designed protein-protein interfaces. In the crystal structure, lattice contacts are formed from the hydrophobic residues intended to form the homodimer interface.Open in a separate windowFig. 3.Crystallographic analysis. (A) Overlay of D_3_212 (PDB: 7RMX) design model (green and cyan) and crystal structure (gray). Top from Left to Right: superposition of the homodimer, potential binding cavity (gray surface) with functionalizable sidechain positions highlighted in blue, superposition of protein–protein interface with sidechain residues shown as sticks (oxygen atoms are red, nitrogen atoms are blue); Bottom from Left to Right: superposition of the monomer, a repeat unit, and a section of the hydrophobic core with sidechain residues shown as sticks. Associated rmsds are indicated. (B) Overlay of design D_3_633 (PDB: 7RKC) design model (green and cyan) and crystal structure (gray); panels are as in (A). (C) D_3_337 design model and crystal structure. Left: overlay of design D_3_337 (PDB: 7RMY) design model (green and cyan) and monomer crystal structure (gray). Middle: designed homodimer interface, with hydrophobic residues shown as sticks and the two chains colored green and cyan. Right: crystal structure showing the central asymmetric unit in gray and its crystal lattice neighbors colored blue, pink, and yellow. The hydrophobic residues which were intended to form the homodimer interface are shown in spheres forming key crystal contacts.Two of the designs, D_3_212 (Fig. 3A) and D_3_633 (Fig. 3B), fold and assemble to the desired dimeric ellipsoidal architecture with central cavities along the axis of symmetry, albeit with some deviations between the experimentally determined structures and design models (Ca rmsd of 2.59 Å and 2.04 Å, respectively). D_3_212 (PDB: 7RMX) is formed from a 224 amino acid-long monomer with helical rise, radius, and curvature of 0.23 Å, 19.6 Å, and 0.73 rad, respectively, that drifted to 1.6 Å, 19.5 Å, and 0.77 rad as the backbone adjusted during subsequent sequence design steps. This near-ideal repeat protein has helix lengths of 19 and 27 amino acids, which was found to be a near optimal combination to create curved repeat proteins. The D_3_212 homodimer has a central cavity with maximum (calculated without sidechains) height and width of 29 Å and 37 Å and a volume of 3,132 ų. In comparison, D_3_633 (PDB: 7RKC) is formed from a 228 amino acid-long monomer with helical rise, radius, and curvature of 0.68 Å, 20.1 Å, and 0.75 rad, respectively, that drifted to 2.2 Å, 22.1 Å, and 0.68 rad during sequence design. The D_3_633 design has repeating helix lengths of 24 and 29 amino acid that surround a central cavity with maximum height and width of 28 Å and 37 Å and a volume of 4,062 ų. In order to test the strength of the protein-protein interface of this structure, We performed a 500× dilution series from 80 μM to 160 nM (see SI Appendix, Fig. S12), and found that it remained 100% dimeric by SEC.     

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.     

The nature of protein folding pathways

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.     

Nature of the protein universe

The protein universe is the set of all proteins of all organisms. Here, all currently known sequences are analyzed in terms of families that have single-domain or multidomain architectures and whether they have a known three-dimensional structure. Growth of new single-domain families is very slow: Almost all growth comes from new multidomain architectures that are combinations of domains characterized by ≈15,000 sequence profiles. Single-domain families are mostly shared by the major groups of organisms, whereas multidomain architectures are specific and account for species diversity. There are known structures for a quarter of the single-domain families, and >70% of all sequences can be partially modeled thanks to their membership in these families.