이 프로젝트는 알프레드 P. 슬로안 재단, NSF 경력 수상 (DBI 1,027,394), 그리고 일반 의료 과학 국립 연구소 (GM083107, GM084222)에 의해 부분적으로 지원됩니다.

<ol>
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위에 제시 프로토콜 구조와 I - TASSER 서버를 사용하여 함수 모델링을위한 일반적인 지침입니다. 이 자동화된 절차는 단백질의 대부분 잘 작동하지만, 인간의 개입은 종종 특히 PDB 라이브러리에 가까운 템플릿 부족한 단백질을 위해, 크게 모델링의 정확도를 개선하는 데 도움이됩니다. (B) 구조 어셈블리를 개선하기 위해 외부 구속을 제공,, 멀티 도메인 단백질 (A) 분리 및 (C) 모델링하는 동안 템플릿을 제거 : 사용자는 다음과 같은 방법으로 I - TASSER 모델링하는 동안 개입하실 수 있습니다. 분할 다중 도메인 단백질 : 오랜 단백질 시퀀스가​​ 자주 실험과 전산 두 기술을 사용하여 구조 해설 어렵게 만드는 유연 링커 지역에 의해 곁에 여러 도메인을 포함합니다. 도메인 독립적으로 엔티티를 접는하고 독특한 분자 기능을 수행할 수있는 그럼에도 불구하고, 그것은이다별도로 각 도메인 긴 다중 도메인 단백질과 모델을 분할하는 것이 바람직하다. 모델링 도메인은 개별적에만 예측 프로세스 속도를하지 않을뿐만 아니라, 쿼리 템플릿 정렬의 품질보다 신뢰할 수있는 구조와 기능 예측의 결과로, 증가합니다. 단백질 시퀀스에서 도메인 경계는 그러한 NCBI CDD 24 PFAM 25 InterProScan 26로 자유롭게 사용할 외부 프로그램을 사용하여 온라인으로 예측할 수 있습니다. LOMETS 스레딩 정렬이 쿼리 단백질에 사용할 수있는 경우 또한, 도메인 경계는 시각 (단계 5.4 참조) 상단 스레딩 템플릿 unaligned 잔류 긴 펼쳐진을 식별하여 찾을 수 있습니다. 이러한 unaligned 지역은 대부분 도메인 링커 지역에 해당합니다. 다중 도메인 템플릿 정렬 모든 쿼리 도메인으로 템플릿 PDB 라이브러리에 이미있는 경우 다음 쿼리 단백질은 전체 길이로 모델 수 있습니다. 외부 구속을 제공 <p클래스 = &quot;jove_content&quot;&gt; I - TASSER의 구조 조립 시뮬레이션은 주로 템플릿을 스레딩 LOMETS에서 수집한 공간적 구속하여 안내하고 있습니다. 템플릿 라이브러리에 좋은 스레딩 히트 (Norm. Z - 점수&gt; 1)이 쿼리 단백질 들어, 파생된 공간 억제는 높은 정확도의 대부분이며, I - TASSER이 단백질에 대한 고해상도 구조 모델을 생성합니다. Contrarily, 약한가 쿼리 단백질 또는 전혀 스레딩 히트 (Norm. Z - 점수 &lt;1), 수집 공간 억제는 종종 때문에 템플릿과 정렬의 불확실성의 오류가 포함되어 있습니다. 이러한 단백질 타겟 들어, 사용자가 지정한 공간 정보는 예측 모델의 품질을 향상시키기 위해 매우 도움이 될 수 있습니다. 사용자는 두 가지 방법으로 I - TASSER 서버로 외부 구속을 제공할 수 있습니다 : A. 연락처 / 거리 제한을 지정 실험적 특징 상호 잔여물 연락처 / 거리, NMR의 예 또는가교 실험을 구속 파일을 업로드하여 지정할 수 있습니다. 예를 들어 파일을 열 1 규제의 종류를 지정합니다 그림 8에 표시됩니다 &quot;DIST&quot;또는 &quot;접촉&quot;즉. 거리 구속에 대한 (DIST), 컬럼 2, 4 잔류 위치를 (I, J)를 포함, 컬럼 3, 5 두 지정한 원자 사이의 거리를 지정 잔여물 및 열 6 원자 유형을 포함합니다. 문의 묶기 위해 (연락처), 컬럼 2, 3 연락해야 잔류물의 위치를 (I, J)가 포함되어 있습니다. 이러한 접촉 잔여 쌍 측면 체인 센터 사이의 거리는 PDB 알려진 구조에서 관찰 거리에 따라 결정됩니다. I - TASSER의 구조 상세 시뮬레이션 동안 지정된 거리에 가까운 이러한 원자 쌍을 그릴려고합니다. B.는 단백질 구조 템플릿을 지정 LOMETS 스레딩 프로그램은 쿼리 제자에 대한 그럴듯한 주름을 찾을 대표 PDB 라이브러리를 사용EIN. 대표적인 구조 라이브러리를 사용하면 시퀀스 구조의 정렬을 계산하는 데 필요한 시간을 줄이기 위해 도움이되지만 그것이하더라도, 그것은 좋은 템플릿 단백질이 도서관에서보고 있거나 템플릿 LOMETS 스레딩 프로그램에서 확인되지 않았을 수도 있습니다 도서관에 존재. 이러한 경우에는 사용자는 템플릿으로 원하는 단백질 구조를 지정해야합니다. 추가 템플릿으로 단백질 구조를 지정하려면, 사용자는 하나 PDB 형식의 구조 파일을 업로드하거나 PDB 라이브러리에 쌓인 단백질 구조의 PDB ID를 지정할 수 있습니다. I - TASSER이 소집 프로그램 23을 사용하여 쿼리 템플릿 정렬을 생성하고 지정된 사용자 템플릿과 구조 조립 시뮬레이션을 안내 LOMETS 템플릿 모두에서 공간적 구속을 수집합니다. LOMETS 묶기의 정확성이 다른 대상에 대해 서로 다른이기 때문에 LOMETS 묶기의 무게는 쉬운 (상동) 타에 강해체계적으로 우리의 벤치 마크 훈련 조정되었습니다 하드 (비 상동) 목표에보다 rgets. 사용자는 자신의 쿼리 템플릿 정렬을 지정할 수 있습니다. FASTA 형식 (그림 9A)와 3D 형식 (그림 9B) : 서버는 두 형식의 정렬을 허용합니다. FASTA 형식은 표준에서 설명 <a href="http://zhanglab.%20ccmb.med.umich.edu/FASTA/">http://zhanglab. ccmb.med.umich.edu / FASTA /</a> . 3D 형식은 표준 PDB 포맷 (유사합니다 <a href="http://www.wwpdb.org/documentation/format32/sect9.html">http://www.wwpdb.org/documentation/format32/sect9.html</a> )하지만 템플릿에서 파생된 두 개의 추가 열이 원자 기록 (그림 9B 참조)에 추가됩니다 : 열 10-30 : 아톰 (단 C - 알파)와 잔여물 질의 순서에 대한 이름입니다. 열 31-54 : 템플릿에 해당 원자에서 복사한 쿼리의 C - 알파 원자 좌표. 열 55-59 : 대응 잔여물 번호를 템플릿에이 배열을 기반으로 열 60-64 : 대응 잔여물 이름 템플릿에 템플릿 단백질을 제외 단백질은 유연한 분자 및 생물 학적 활동을 변경하는 여러 conformational 상태를 채택할 수 있습니다. 예를 들어, 많은 단백질 kinases 및 막 단백질의 구조 활성 및 비활성 두 형태에서 해결되었습니다. 또한 행 리간드의 존재 또는 부재는 큰 구조적 움직임을 일으킬 수 있습니다. 템플릿의 모든 conformational 상태는 스레딩 프로그램에 똑같지만, 그것은 하나의 특정 상태에있는 템플릿을 사용하여 쿼리를 모델하는 것이 바람직하다. 서버에 새로운 옵션은 사용자가 구조를 모델링하는 동안 템플릿 단백질을 제외 수 있습니다. 이 기능은 또한 사용자가 모델링에 사용되는 템플릿의 상동 수준을 선택할 수 있도록합니다. 사용자는 템플릿 단백질 천을 제외시킬 수 있습니다톰 I - TASSER 도서관으로 : A.이 시퀀스 신분 컷오프 지정 사용자는 I - TASSER 템플릿 라이브러리에서 상동 단백질을 제외하려면이 옵션을 사용할 수 있습니다. 상동 수준이 시퀀스 신분 컷오프에 따라 설정, 쿼리 및 쿼리 순서의 시퀀스 길이로 나눈 템플릿 단백질 사이의 동일 물질의 수를 즉. 예를 들어, 제공되는 양식에 &quot;70%&quot;의 사용자 유형,&gt; 70 %를 쿼리 단백질 내가 - 것입니다 I - TASSER 템플릿 라이브러리에서 제외 될 수있는 시퀀스 ID를 가진 모든 템플릿 단백질. B. 특정 템플릿 단백질을 제외 특정 템플릿 단백질은 제외하는 구조의 PDB ID를 포함하는 목록을 업로드하여 I - TASSER 템플릿 라이브러리에서 제외시킬 수 있습니다. 예제 파일은 그림 10에 표시됩니다. 같은 단백질은 PDB 라이브러리에서 여러 항목, I - TASSER SE로 존재 수 있듯이rver는 기본적으로 지정된 템플릿 (COLUMN1에서)뿐만 아니라 평범한 생활을 도서관에서 다른 모든 템플릿&gt; 지정된 템플릿 90 %를 제외합니다. 사용자는 ID를 모든 템플릿&gt; 지정된 템플릿 단백질 70 %가 제외됩니다 다른 신분 컷오프, 예를 들어 70 %, 지정할 수 있습니다.

<ol><li> FASTA는 (참조 모델로 단백질의 아미노산 서열 포맷 <a href="http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml">http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml을</a> ). </li><li> 인터넷에 접속하고 웹 브라우저와 함께 개인용 컴퓨터. </li><li> 예측 차 구조와 기능 사이트를 분석하는 분자 시각화 소프트웨어, 예를 들어 RASMOL 또는 PYMOL. </li></ol>

a protocol for computer-based protein structure and function prediction

게놈 시퀀싱 프로젝트는 생물 학적 역할에 대한 이해를 향상시키기 위해 자신의 구조와 기능에 대한 지식을 필요로 단백질 시퀀스의 수백만을 계산합니다. 실험 방법이 단백질의 작은 일부분에 대한 자세한 정보를 제공할 수 있지만, 전산 모델링을 실험적으로 uncharacterized 아르 단백질 분자의 대부분 필요합니다. I - TASSER 서버는 단백질 구조와 기능의 고해상도 모델링을위한 온라인 작업대입니다. 단백질 시퀀스 감안할 때, I - TASSER 서버에서 전형적인 출력, 보조 구조 예측을 포함 각 물질의 용매 접근성, 스레딩 및 구조 정렬에 의해 감지 동종 템플릿 단백질, 최대 다섯 전체 길이 차 구조 모델 및 구조 기반을 예측 효소 분류에 대한 주석 기능, 유전자 온톨로지 조건과 단백질 - 리간드 결합 사이트. 모든 예측은 신뢰 점수 태그가있는예측은 실험 데이터를 모르고하는 방법 정확하게 알려줍니다. 최종 사용자의 특별한 요청을 촉진하기 위해서, 서버는 사용자가 지정한 간 잔여 거리를 수락하고 대화형 I - TASSER 모델링을 변경하려면지도를 연락 채널을 제공하고, 또한 사용자가 템플릿으로 어떤 단백질을 지정할 수 있습니다, 또는 서식 파일을 제외 구조 조립 시뮬레이션 중에 단백질. 구조 정보는 I - TASSER 예측의 품질을 향상 목적으로 실험 증거 또는 생물 학적 지식을 바탕으로 사용자가 수집한 수 있습니다. 서버가 최근 사회 전체 CASP 실험에서 최고의 단백질 구조에 대한 프로그램 및 기능 예측으로 평가되었다. 온라인 I - TASSER 서버를 사용하는 100 개 이상의 나라에서&gt; 20000 등록 과학자들은 현재이 있습니다.

Watch this Scientific Journal Video about A Protocol for Computer-Based Protein Structure and Function Prediction at JoVE.com

A Protocol for Computer-Based Protein Structure and Function Prediction

I - TASSER 파이프라인을 사용하여 단백질의 구조와 기능 특성화 기반 컴퓨터에 대한 지침이 설명되어 있습니다. 쿼리 단백질 시퀀스에서 시작, 3D 모델은 여러 스레딩 정렬을 사용하여 생성 및 구조 조립 시뮬레이션을 반복하고 있습니다. 기능성 inferences는 이후 알려진 구조와 기능을 가진 단백질 일치를 기반으로 그려져있다.

컴퓨터 기반의 단백질 구조 및 기능 예측을위한 프로토콜

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

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Biology

68.1K 조회수.  University of Michigan. I - TASSER 파이프라인을 사용하여 단백질의 구조와 기능 특성화 기반 컴퓨터에 대한 지침이 설명되어 있습니다. 쿼리 단백질 시퀀스에서 시작, 3D 모델은 여러 스레딩 정렬을 사용하여 생성 및 구조 조립 시뮬레이션을 반복하고 있습니다. 기능성 inferences는 이후 알려진 구조와 기능을 가진 단백질 일치를 기반으로 그려져있다.

비디오: A Protocol for Computer-Based Protein Structure and Function Prediction

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

동력학 및 구조 기능 분석의 선호도 결정에 대한 올바른 단백질 농도의 중요성

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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생물학

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

버딩 효모에서 단백질 농도의 함수로 형태학의 표현형의 발전 분석

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

을 바탕으로 단백질 - 단백질 상호 작용의 확인에 프로토콜<sup&gt; 15</sup&gt; N 신진 대사 라벨링, Immunoprecipitation, 수량 질량 분석법 및 선호도 변조

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

재조합 단백질의 미끼를 사용 파지 디스플레이와 궁합 선택을위한 프로토콜

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

단백질 상호 작용을 발견하고 HaloTag 기술을 사용하여 단백질의 기능을 특성화

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

조사 단백질 기능, 시공간 현지화 및 단백질 상호 작용 네트워크를위한 유도 LAP-태그 안정 세포주

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...