Nonenzymatic Protein Function And Protein Analysis - Ch. 3 Flashcards
Collagen
Structural protein - characteristic trihelical fiber makes up most of the extracellular matrix of connective tissue. Deficiency causes brittle bone disease
Elastin
Structural protein - another component of extracellular matrix of connective tissue; it stretches and recoils like a spring
Keratins
Structural protein - intermediate filament proteins found in epithelial cells
Contribute to mechanical integrity of cell and function as regulatory proteins (hair and nails)
Actin
Structural protein - protein that makes up microfilaments and thin filaments in myofibrils.
Most abundant protein in eukaryotic cells
Positive side and negative side polarity allows motor proteins to travel unidirectional along an actin filament
Tubulin
Structural protein- makes up microtubules important for providing structure, chromosomal separation in mitosis and meiosis and intercellular transport of kinesin and dynein
Motor proteins
Some interact with structural proteins
Display enzymatic activity acting as ATPases that power conformational change
Transient interactions with either actin or microtubules
Myosin
Motor protein - primary one that interacts with actin
Thick filament in myofibril and involved in cellular transport sometimes
Kinesins and dyneins
Motor proteins- associated with microtubules
They have two heads, one of which is attached to tubulin at all times
Kinesins - role in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis - move vesicles to positive end of cell
Dyneins - involved in the sliding movement of cilia and flagella- move vesicles to negative end of cell
Binding proteins
Stabilizing functions in individual cells and body
Include hemoglobin, calcium-binding proteins, DNA-binding proteins
Has affinity for its molecule of interest
Cell Adhesion molecules
CAMs
Found on surface of most cells and aid in binding the cell to the extracellular matrix of other cells
Cadherins
Family of CAMs
Group of glycoproteins that mediate calcium-dependent cell adhesion
Often hold similar cell types together
Different cells - different types of cadherins
Integrins
Family of CAMs
Group of proteins that have two membrane spanning chains called alpha and beta important for binding to and communicating with extracellular matrix
Promote cell division, apoptosis and other processes
Selectins
Family of CAMs
Group of proteins that bind to carb molecules that project from other cell surfaces
Weaker bonds formed by CAMs than cadherins and integrins
Expressed on WBCs and endothelial cells that line blood vessels
Important in host defense
Immunoglobulins
Also called antibodies or immunoglobulins (Ig)
Most prominent type of protein found in immune system
Produced by B cells target threats and recruit cells to help
Y-shaped made of two identical heavy chains and two identical light chains held together by disulfide linkages and noncovalent interactions
Antigen binding region
Area of Ig (immunoglobulins or antibodies) in which there is a specific polypeptide sequence that will bind only one specific antigenic sequence
Possible outcomes of immunoglobulins binding with antigens
- Neutralizing antigen
- Marking pathogen for destruction (called opsonization)
- clumping together (agglutinating) the antigen so it can be phagocytized
Biosignaling
Process by which cells receive and act on signals
Ion channels
Proteins that create specific pathways for charged molecules
Facilitated diffusion
Ion channels
A type of passive transport - diffusion of molecules down a concentration gradient through a pier in the membrane
Ungated ion channel
Unregulated; example potassium channels.
Voltage-gate ion channels
Regulated by membrane potential change near channel
Membrane depolarization can cause conformation change to make them quickly open and quickly close again (sinoatrial node in heart is example - pacemaker)
Ligand-gated ion channels
Binding of specific substance or ligand to channel causes it to open or close
Enzyme-linked receptors
Catalytic activity in response to ligand binding
Three primary protein domains: membrane-spanning domain, catalytic domain, and ligand-binding domain
Membrane-spanning domain
Enzyme linked receptor that anchors the receptor in the cell membrane
Ligand-binding domain
Enzyme linked receptor that is stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain
G-Protein Coupled Receptors
GPCR
Large family of integral membrane proteins involved in signal transduction
G-Proteins
Named for intercellular link to guanine nucleotides (GDP and GTP)
Three main types: Gs which stimulates adenylate cyclase increases levels of cAMP in cell
Gi inhibits and decreases levels of cAMP
Gq activates phospholipase C
Subunit of G protein
Alpha binds GDP and in in complex with beta and gamma subunits
Protein isolation
Means of separating protein of interest for study
Isolated by cell lysis and homogenization - the act of crushing, grinding, or blending the tissue of interest into an evenly mixed solution.
Centrifugation - then isolates proteins from smaller molecules
Electrophoresis
Electric field with positively charged anode end which attracts anions and negatively charged cathode end attracts cations
Polyacrylamide gel is the standard medium
Migration velocity equation
In electrophoresis, migration velocity (v) of molecule is equal to electric field strength (E) times net charge on molecule (z) both over frictional coefficient (f) which depends on mass and shape
Native PAGE
Polyacrylamide gel electrophoresis - analyzation of proteins in their native states
Drawbacks: limited by varying mass-to-charge and mass-to-size ratios of cellular protein
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis- separates proteins on the basis of mass alone. Starts with PAGE but adds SDS, a detergent that disrupts all noncovalent interactions
Denatures protein
The only variables affecting velocity are E and f
Isoelectric focusing
Electrophoresis separating proteins based on their isoelectric point (pI) - pH at which it is electrically neutral
Based on acidic and basic properties - placed in gel with pH gradient acidic on positive anode and basic gel at negative cathode, neutral in middle.
Movement towards what it needs will cause the pH to equal the pI and the protein to stop migrating.
Chromatography
The more similar a compound is to its surroundings (by polarity, charge etc) the more it will stick to and move slowly through them
First sample onto solid medium called stationary phase or adsorbent
Then run motile phase through stationary phase allowing it to elute - affinity for this phase results in fast migration
Retention time
Amount of time substance spends in stationary phase in chromatography
Partitioning
Separation of components in stationary phase
Column chromatography
Column filled with silica or alumina beads as adsorbent
Gravity moves solvent and compounds down the column
Polarity and size effect how fast. The less polar the faster it will move.
Salinity can easily be changed to help elute protein of interest
Ion-exchange chromatography
Beads in silica and alumina in column are coated with charged substances so they attract and bind substances of opposite charge
Size-exclusion chromatography
Beads in column contain tiny pores and keep small compounds and allow bigger ones to elute
Affinity chromatography
Customized columns with high affinity for a particular protein by coating beads with receptor that binds that protein or specific antibody for that protein
Protein is retained
X Ray crystallography
Isolated and crystallized protein beforehand
Most reliable and common
Diffraction pattern of small dots can be interpreted to determine protein’s structure
NMR Spectroscopy
Nuclear magnetic resonance - accounts for 25% of structural determination
Edman degradation
Uses cleavage to sequence proteins of up to 50 to 70 amino acids. Selectively and sequentially removes the n-terminus amino acid of the protein
UV Spectroscopy
Proteins have aromatic side chains and can be analyzed with this without treatment. But this is sensitive to contamination.
Bradford protein assay
Mixes a protein in solution with Coomassie brilliant blue dye
Protonated, dye is brown-green
