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Amino Acid Sequence: The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining Protein conformation.

Protein Folding

JoVE 10679

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation which is critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.

Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing immunological defense, storage, transport, cellular communication, movement, and structural support. A protein’s function mostly depends on its ability to recognize and bind other molecules, analogous to a lock and key. Hence the specific activity of each protein depends on its unique three-dimensional architecture. For a protein to be functional, it must fold accurately. Most proteins go through several intermediate forms before folding into the most stable, biologically active structure. Misfolding of proteins has detrimental effects on the overall functioning of the cell. In humans, several diseases are due to the accumulation of misfolded or unfolded proteins. These include cystic fibrosis, Alzheimer’s, Parkinson’s, ALS, and Creutzfeldt-Jakob disease. Proteins are made up of one or more chains of amino acids, called polypeptides. A polypeptide is synthesized as a linear chain which rapid

 Core: Macromolecules

Mutations

JoVE 10793

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.

Mutations that occur at a single nucleotide are called point mutations. When point mutations occur within genes, the consequences can vary in severity depending on what happens to the encoded amino acid sequence. A silent mutation does not change the amino acid identity and will have no effect on an organism. A missense mutation changes a single amino acid, and the effects might be serious if the change alters the function of the protein. A nonsense mutation produces a stop codon that truncates the protein, likely rendering it nonfunctional. Frameshift mutations occur when one or more nucleotides are inserted into or deleted from a protein-coding DNA sequence, affecting all of the codons downstream of the location of the mutation. The most drastic type of mutation, chromosomal alteration, changes the physical structure of a chromosome. Chromosomal alterations can include deletion, duplication, or inversion of large stretches of DNA within a single chromosome, or integration o

 Core: DNA Structure and Function

Types of RNA

JoVE 10800

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use. The central dogma of molecular biology states that DNA contains the information that encodes proteins and RNA uses this information to direct protein synthesis. Different types of RNA are involved in protein synthesis. Based on whether or not they encode proteins, RNA is broadly classified as protein-coding or non-coding RNA. Messenger RNA (mRNA) is the protein-coding RNA. It consists of codons—sequences of three nucleotides that encode a specific amino acid. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNA. tRNA acts as an adaptor molecule that reads the mRNA sequence and places amino acids in the correct order in the growing polypeptide chain. rRNA and other proteins make up the ribosome—the seat of protein synthesis in the cell. During translation, ribosomes move along an mRNA strand where they stabilize the binding of tRNA molecules and catalyze the for

 Core: Gene Expression

Complementary DNA

JoVE 10818

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene. cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away. Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a pol

 Core: Biotechnology

Electrospinning of Silk Biomaterials

JoVE 5798

Silk fibers have been processed and used to create fabrics and threads for centuries. However, the solubilizing of silk fibers, thereby turning it into a versatile pre-polymer solution is a much newer technology. Solubilized silk can be processed in many different ways to create a biocompatible material with controllable mechanical properties.


 Bioengineering

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

1Department of Neurobiology, F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, 2Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, 3Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, 4Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research

JoVE 57633

 Genetics

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

1Division of Nephrology and Hypertension, and the Center for Kidney Research and Therapeutics at the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, 2Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, 3School of Biological Sciences and Medical Engineering, Southeast University

JoVE 56278

 Biochemistry

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

1Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, 2Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences

JoVE 3657

 Immunology and Infection

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

1Dipartimento di Biotecnologie, Università degli Studi di Verona, 2Ecology and Evolution, Research School of Biology, The Australian National University, 3Dipartimento di Agraria, SACEG, Università degli Studi di Sassari

JoVE 56626

 Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

JoVE 54093

 Biology

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

1Department of Anesthesiology, Medical College of Wisconsin, 2Clement J. Zablocki Veterans Affairs Medical Center, 3Department of Surgery, Division of Pediatric Surgery, Children's Research Institute, 4Department of Orthopedic Surgery, Medical College of Wisconsin, 5Deptarment of Anesthesiology, Clement J Zblocki Veteran Affairs Medical Center, 6Department of Medicine, Division of Cardiology, Medical College of Wisconsin

JoVE 55036

 Immunology and Infection

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

1Protein Crystallization Lab, Emerald Bio, 2Molecular Biology Lab, Emerald Bio, 3Scientific Sales Representative, Emerald Bio, 4Group Leader II, Emerald Bio, 5Group Leader I, Emerald Bio, 6Chair of Advisory Board, Emerald Bio, 7Director of Multi-Target Services, Emerald Bio, 8Senior Project Leader, Emerald Bio, 9Project Leader II & SSGCID Site Manager, Emerald Bio

JoVE 4225

 Immunology and Infection

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4The State Key Laboratory of Medical Genetics, Central South University

JoVE 56739

 Cancer Research

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation

JoVE 52610

 Immunology and Infection
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