Chapter 3: Nonenzymatic Protein Function and Protein Analysis

Biochemistry Chapter 3

Types of Structural Proteins

Collagen, Elastin, Keratin, Actin and Tubulin

Collagen

trihelical fiber and makes up most of extracellular matrix of connective tissue, found throughout body and important in strength and flexibility

Elastin

Component of extracellular matrix of connective tissue, role to stretch then recoil which restores original shape of tissues

Keratins

Intermediate filament proteins found in epithelial cells. Contribute to mechanical integrity of cell and also function as regulatory proteins

Actin

Make up microfilaments and thin filaments in myofibrils, most abundant protein in eukaryotic cells

Tubulin

Protein makes up microtubulus

Myosin

Primary motor protein that interacts with actin, involved in cellular transport

Kinesins and dyneins

motor proteins associated with microtubules

Kinesin

play role in alligning chromosome during metaphase and depolymerizing microtubule during anaphase mitosis

Dyneins

Sliding movement cilia and flagella

Binding Proteins

Hemoglobin, Ca 2+ binding proteins and DNA binding proteins

Cell Adhesion molecules

proteins found on surface of most cells and aid inbinding the cell to the extracellular matrix or other cells

Cadherins

Group of glycoproteins that mediate calcium dependent cell adhesion

Integrins

Group of proteins that all have two membrane spanning chains called a and b

Selectins

Bind to carbohydrate molecules project from other cell surfaces

Antibodies

proteins produced by beta cells that function to neutralize targets in the body and then recruit other cells eliminate threat

Biosignaling

process cells recieve and act on signals

Ion channels

proteins that create specific pathways for charged molecule

Facilitated diffusion

Diffusion of molecules down a concentration gradient through a pore in the membrane created by trans membrane protien

Ungated channels

have no gates and therefore unregulated

Voltage gated channels

Gate regulated by membrane potential change near the channel

Ligand gated channels

binding specpfic substance or ligand to channel causes it to open or close

Membrane spanning domain

anchor receptor in cell membrane

Ligand binding domain

stimulated by appropriate ligand nad induces conformational change activates catalytic domain

GPCR

large family of integral membrane proteins involved in signal transduction

G3

stimulates adenylate cyclase increasing cAMP in cells

G1

inhibits adenylate cyclase decreasing cAMP in cells

Gq

activates phospholipase C

Electrophoresis

Works by subjecting compounds to an electric field where it moves according to net charge and size

Polyacrylamide gel

Standard medium for protein electrophoresis

Polyacrylamide gel electrophoresis PAGE

Method to analyze proteins in native states

Native PAGE

maintains proteins shape, but results are difficult to compare because the mass to charge ratio differs for each protein

SDS PAGE

denature the proteins and mask the native charge so comparison of size is more accurate, functional protein cannot be recapture from gel

Reducing PAGE

All non covalent interaction are preserved, but reducing agent will break any disulfide links in tertiary and quaternary structure reducing to cysteins

Isoelectric point

The pH at which the protein or amino acid is electrically neutral,

Isoelectric focusing

exploits the acidic and basic properties fo amino acids by separating on basis of pI

Chromatography

another tool uses physical and chemical properties to separate and identify compound from complex mixture

retention time

amount fo time compound spends in stationary phase

Column Chromatography

column filled with silica or alumnia beads gravity moves solvent and compounds down columns

Ion exchange method

beads in column are coated with charged substances so they attract or bind compounds that have opposite charge

Size exclusion chromatography

Beads used in column contain tiny pores of varying sizes

Affinity chromatography

Uses a bound receptor or ligand and an eluent with free ligand or receptor for protein of interest

Edman degradation

Cleavage sequence proteins up to 50-70 amino acids. Selectively and sequentially removes the N terminal amino acid of the protein

Enzyme Acitvity

measures as moles of substrate converted by the enzyme into pot per unit of time

Specific activity

Measures activity of the enzyme per mass total protein concentration.