Unnatural Amino Acids as Probes of Protein Structure and Function

针对病毒进入和成熟的新型药物最近被添加到不断增长的抗病毒药物军械库中，旨在阻止HIV感染的持续传播。此外，参与HIV复制关键步骤的宿主因子正在被考虑作为治疗干预的靶点。尽管有这些有希望的方法，病毒酶PR、RT和IN仍然是高活性抗逆转录病毒治疗的主要目标，有近20种药物用于对抗PR和RT，而IN抑制剂正在进行临床试验。关于HIV-1 RT，我们将生物化学和生物物理学与结构、分子和化学生物学相结合，以提供其在将入侵病毒的RNA转化为整合能力双链DNA中的作用的分子细节。利用核苷和氨基酸类似物对代表复制中间体的复合物结构提供新的敏锐度，补充了传统的核酸和蛋白质诱变方法。化学核酸足迹现在与直接质谱分析相辅相成，而单分子荧光研究已经被引入来探测各种核酸双链上RT的取向。通过翻译抑制将具有新型生物物理特性的非天然氨基酸引入蛋白质中，从而实现具有极高分辨率的结构/功能分析。我们之前的研究集中在分析HIV-1 RT功能对具有不同生物物理性质的非天然氨基酸靶向插入的响应。我们现在正在通过开发研究病毒和宿主蛋白相互作用的方法，将这项技术扩展到更多的生物学背景中。Ganser等人报道了HIV-1 CA-NC融合蛋白在RNA存在下组装成在形态上类似于真正核心的结构的能力（Science 1999）。随后，CA-NC管被用于了解宿主因子介导的核分离限制。利用Chin等（PNAS 2002）开发的翻译抑制技术，将对苯甲酰-l-苯丙氨酸（Bpa）位点特异性插入到大肠杆菌中表达的重组CA- nc管的CA的几个位置。在RNA的存在下，这些突变体组装成光活性管，这是对未修饰结构的合理模仿。与免疫沉淀等传统方法相比，光交联的优势在于它可以立即识别CA-NC上的结合区域，并通过质谱加速识别结合伙伴及其接触点。此外，弱和瞬态相互作用更容易被光交联检测到。作为原理证明，我们将在宿主蛋白如亲环蛋白和CPSF6的已知相互作用位点插入Bpa。之后，含bpa的CA-NC管将与细胞裂解液一起孵育，并使用免疫沉淀和质谱相结合的方法照射以寻找其他宿主限制因子。该项目将与Vineet KewalRamani博士合作进行，他的团队将提供细胞培养设施和培训。虽然我们可以重建含有Bpa的CA-NC管，这些管在形态上合理地模仿了野生型结构，但这种方法存在潜在的缺陷：(1)Bpa插入虽然具有位点特异性，但可能诱导构象变化，抑制宿主因子结合；(2) Bpa插入位点本身可能与结合位点重叠；(3) Bpa的插入可能优先诱导交联CA-NC二聚体。因此，我们提出了一种利用光活性氨基酸光亮氨酸和光亮氨酸的互补策略。这些含有重氮嘧啶的氨基酸与天然氨基酸的相似性克服了真核细胞的身份控制元件，使它们能够通过未经修饰的翻译机制被纳入蛋白质中。Photo-Leu和photo-Met是市售的，已被其他研究小组用于检测活细胞中的蛋白质相互作用。因此，我们的策略将是在缺乏亮氨酸和蛋氨酸但含有其光活性对应物的培养基中培养适当的真核细胞系。这些细胞裂解液将在野生型CA-NC管中孵育，并在365 nm处照射以确定相互作用的伙伴。CA-NC管将通过免疫沉淀法或速率、带沉降法回收，并通过质谱法分析相互作用的伙伴和相互作用的部位。尽管付出了巨大的努力，我们仍未获得HIV-1 RT的含葡萄状尿素共晶。尽管具有增加葡萄状尿素敏感性的HIV-1 RT突变体或相关慢病毒酶（EIAV）可能促进结晶，但我们已经实施了一种替代溶液策略，以提供抑制剂结合后葡萄状尿素结合位点附近构象变化的信息。该策略利用氟化Phe衍生物OCF3-Phe的位点特异性结合。我们将p66残基Tyr501替换为OCF3-Phe，没有损失DNA聚合酶和RNase H活性，并在酶浓度为50 uM时获得突变体的19F谱。实验上，这需要纯化3毫克重组的选择性突变的HIV-1 RT，这在我们的能力范围内。将制备p51 thumb（如Trp266、Tyr271、Tyr318、Tyr319）或催化残基附近p66 RNase H结构域（如Tyr441、Tyr532、Tyr483）中含有芳香-&gt； OCF3-Phe取代的突变酶，分析DNA聚合酶和RNase H活性的保留情况，并在无抑制剂和存在抑制剂的情况下通过19F-NMR进行检测。我们将与Angela Gronenborn博士合作进行核磁共振研究，其中19F冷冻探针可提供10-20 μ m蛋白质浓度下的光谱。我们在这个项目中追求的另一个策略是与ZIA BC 011443项目的单分子光谱研究相结合，其目标是保留p66和p51 HIV-1 RT （Cys38和Cys280）的天然半胱氨酸，并引入Phe-&gt；叠氮-Phe （Az-Phe）取代，以引入烷基基荧光团（Kolb等人，Angew）。化学。Int。英文版。2001)。考虑到RT中芳香残基的丰度，这种方法比靶向Cys插入到不含Cys的酶中具有更大的灵活性。其次，我们建议将单分子光谱研究扩展到包括其他慢病毒和γ -抗逆转录病毒RT，其中消除大量半胱氨酸（MLV/XMRV RT为8个，EIAV RT为7个）变得不切实际。作为一种替代策略，Az-Phe将通过Click化学通过翻译抑制炔基Cy3或Cy5的附着，特异性地引入p66或p51 RT中，这涉及叠氮化物和炔功能的偶联，以产生1,2,3-三唑。NIH Image Probe Development Center的Gary Griffiths博士为我们合成了炔基Cy3和炔基Cy5。作为原理证明，我们将在p66的N端或c端引入Az-Phe，并比较单标记RT与cys标记RT的构象动力学，我们有相当多的数据。这一策略开启了建立三色FRET的可能性，每个RT亚基上有荧光团，核酸底物上有第三个荧光团，以检查伴随其核酸易位的酶内的构象变化，类似于Lee等人对大肠杆菌RNA聚合酶的研究。j . 2007)。[对应2011年10月HIV耐药项目实地考察报告中的Le Grice Project 3]

Novel drugs targeting virus entry and maturation have recently been added to the growing armory of antiviral agents aimed at stemming the continuing spread of HIV infection. In addition, host factors participating in key steps in HIV replication are under consideration as targets for therapeutic intervention. Despite these promising approaches, the viral enzymes PR, RT, and IN remain primary targets of highly active antiretroviral therapy, with almost 20 drugs in use against PR and RT, and IN inhibitors undergoing clinical trials. With respect to HIV-1 RT, our section combines biochemistry and biophysics with structural, molecular, and chemical biology to provide molecular details of its role in converting RNA of the invading virus into integration-competent double-stranded DNA. Using nucleoside and amino acid analogs to provide novel acumen into the structures of complexes representing replication intermediates complements traditional nucleic acid and protein mutagenesis methods. Chemical nucleic acid footprinting is now complemented with direct mass spectrometric analysis, while single-molecule fluorescence studies have been introduced to probe the orientation of RT on a variety of nucleic acid duplexes. Site-specific introduction of unnatural amino acids with novel biophysical properties into proteins by translational suppression permits a structure/function analysis with an exceptionally high level of resolution. Our previous studies concentrated on analyzing HIV-1 RT function in response to targeted insertion of unnatural amino acids with different biophysical properties. We are now extending this technology into a more biological context by developing methods to study the interaction of viral and host proteins. The ability of an HIV-1 CA-NC fusion protein to assemble, in the presence of RNA, into structures that morphologically resemble authentic cores was reported by Ganser et al. (Science 1999). Subsequently, CA-NC tubes have been used to understand host factor-mediated restriction of core dissembly. Using the translational suppression technology developed by Chin et al. (PNAS 2002), p-benzoyl-l-phenylalanine (Bpa) was site-specifically inserted into CA at several positions of recombinant CA-NC tubes expressed in E. coli. In the presence of RNA, these mutants assemble into photoactive tubes that are reasonable mimics of unmodified structures. In contrast to traditional methods such as immunoprecipitation, photocrosslinking has the advantage that it immediately identifies the binding region on CA-NC and accelerates identification of the binding partner and its point of contact by mass spectrometry. In addition, weak and transient interactions are more likely to be detected by photocrosslinking. As proof-of-principle, we will insert Bpa at known interaction sites for host proteins such as cyclophilin and CPSF6. Thereafter, Bpa-containing CA-NC tubes will be incubated with cell lysates and irradiated to search for additional host restriction factors using a combination of immunoprecipitation and mass spectrometry. This project will be performed in collaboration with Dr. Vineet KewalRamani, whose group will provide cell culture facilities and training. Although we can reconstitute Bpa-containing CA-NC tubes that are reasonable morphological mimics of the wild-type structure, there are potential pitfalls to this approach: (1) Bpa insertion, while site specific, may induce a conformational change that inhibits host factor binding; (2) the site of Bpa insertion may itself overlap with a binding site; and (3) Bpa insertion may preferentially induce crosslinked CA-NC dimers. We therefore propose a complementary strategy that exploits the photoactive amino acids photo-Leu and photo-Met. Similarity of these diazirine-containing amino acids to their natural counterparts overcomes identity control elements of the eukaryotic cell, allowing them to be incorporated into proteins by the unmodified translational machinery. Photo-Leu and photo-Met, which are commercially available, have been used by other groups to examine protein:protein interactions in living cells. Our strategy will therefore be to culture the appropriate eukaryotic cell lines in medium depleted of Leu and Met but containing their photoactive counterparts. These cell lysates will be incubated with wild-type CA-NC tubes and irradiated at 365 nm to identify interacting partners. CA-NC tubes will be recovered either by immunoprecipitation or rate, zone sedimentation and analyzed by mass spectrometry for the interacting partner and site of interaction. Despite significant effort, we have not obtained vinylogous urea-containing cocrystals of HIV-1 RT. Although HIV-1 RT mutants or related lentiviral enzymes (EIAV) with increased vinylogous urea sensitivity may facilitate crystallization, we have implemented an alternative solution strategy to provide information on conformational changes in the immediate vicinity of the vinylogous urea binding site following inhibitor binding. This strategy exploits site-specific incorporation of the fluorinated Phe derivative OCF3-Phe. We replaced p66 residue Tyr501 with OCF3-Phe without loss of DNA polymerase and RNase H activity and obtained the 19F spectrum of the mutant at an enzyme concentration of 50 uM. Experimentally, this required purifying 3 mg of reconstituted, selectively mutated HIV-1 RT, which is well within our capacity. Mutant enzymes containing aromatic -> OCF3-Phe substitutions in the p51 thumb (e.g., Trp266, Tyr271, Tyr318, Tyr319) or the p66 RNase H domain in the vicinity of catalytic residues (e.g., Tyr441, Tyr532, Tyr483) will be prepared, analyzed for retention of DNA polymerase and RNase H activity, and examined by 19F-NMR in the absence and presence of inhibitor. We will perform NMR studies in collaboration with Dr. Angela Gronenborn, where a 19F cryoprobe that provides spectra at protein concentrations of 10-20 uM is available. Another strategy that we are pursuing in this project is designed in conjunction with single-molecule spectroscopy studies of Project ZIA BC 011443, the goal of which is to retain the natural cysteines of p66 and p51 HIV-1 RT (Cys38 and Cys280) and introduce Phe->azido-Phe (Az-Phe) substitutions in order to introduce an alkynyl-fluorophore (Kolb et al., Angew. Chem. Int. Ed. Engl. 2001). Given the abundance of aromatic residues in RT, this approach gives considerably more flexibility than targeted Cys insertions into a Cys-free enzyme. Secondly, we propose extending single-molecule spectroscopy studies to include other lentiviral and gammaretroviral RTs, where elimination of a significantly larger number of cysteines (8 in MLV/XMRV RT, 7 in EIAV RT) becomes impractical. As an alternative strategy Az-Phe will be site-specifically introduced into p66 or p51 RT via translational suppression for attachment of either alkynyl Cy3 or Cy5 by Click chemistry, which involves coupling of azide and alkyne functions to produce a 1,2,3-triazole. In response to a call for proposals, alkynyl Cy3 and alkynyl Cy5 were synthesized for us by Dr. Gary Griffiths, Image Probe Development Center, NIH. As proof-of-principle, we will introduce Az-Phe at the N- or C-terminus of p66 and compare conformational dynamics of the singly labeled RT with the Cys-labeled counterpart, for which we have considerable data. This strategy opens the possibility of establishing three-color FRET, with fluorophores on each RT subunit and a third on the nucleic acid substrate, to examine conformational changes within the enzyme that accompany translocation on its nucleic acid, akin to studies performed with E. coli RNA polymerase by Lee et al. (Biophys. J. 2007). [Corresponds to Le Grice Project 3 in the October 2011 site visit report of the HIV Drug Resistance Program]