Cytoskeletal Proteins
Structural proteins, anchoring proteins, and a lot of extracellular matrix
* Fibrous with repeating domains
* Function in cellular motility
Collagen, elastin, keratin, actin, tubulin
Motor Proteins
One or more heads able to generate force through a conformational change
* Have ATPase activity and binding heads (indicates catalytic activity)
* Function in cellular motility (nonenzymatic)
* Have enzymes (protein/RNA molecules with catalytic activity)
Myosin, kinesis, dynein
Binding protein
Bind to specific substrate either to sequester in body/hold its concentration at steady state
* If present in high amounts relative to substrate, almost all substrate will be bound despite low affinity
Cadherins
Calcium-dependant glycoproteins that hold similar cells together
Integrins
Two membrane-spanning chains
* Allow cells to adhere to proteins in extracellular matrix
* Some have signaling capabilities
Selectins
Allow cells to adhere to carbohydrates on surfaces of other cells
* Most commonly used in immune system
Antibody to Antigen Bonding
- Neutralization of pathogen/toxin
- Opsonization (marking) of antigen for destruction
- Creation of insoluble antigen-antibody complexes that can be phagocytized and digested by macrophages (agglutination)
Enzyme-linked Receptors
Participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades
* Autoactivity
* Enzymatic activity
G Protein-Coupled Receptors
New membrane-bound protein associated with trimeric G protein
* Initiate second messenger systems
* Two-protein complex
* Dissociation upon activation
* Trimer
Receptor Similarities
- Extracellular domain
- Transmembrane domain
- Ligand binding
Ungated Channels
Ion channels that are always open
* Responsible for maintaining resting membrane potential
* Involved in cell signaling and pacemaker potentials but cause deviation from resting membrane potential
Transport Kinetics
- Display Km and vmax values
- Can be cooperative like some binding proteins
- No Keq values for reactions because no catalysis
Isoelectric Focusing & Ion-Exchange Chromatography
- Seperate proteins based on charge
- Charge of protein in any environment is determined by isoelectric point (pI)
- Isoelectric focusing uses gel with pH gradient that promotes variable charge
Native PAGE
Complete protein recovered after analysis
* More accurately finds relative globular size of proteins
* Maintains protein’s shape
* Results are difficult to compare since mass-to-charge ratio differs for each protein
SDS-PAGE
Denature proteins and masks native charge so size comparison is more accurate
* Can be used to eliminate conflation from mass-to-charge ratios
* Functional protein can’t be recaptured from gel
Sodium dodecy sulfate (SDS) detergent that will digest proteins to form micelles with uniform negative charges
Affinity Chromatography
Uses bound receptor/ligand and eluent with free ligand/receptor for protein of interest
Potential Drawbacks:
1. Protein of interest may not elute from column because its affinity is too high
2. Permanently bound to free receptor in eluent
Inactivations:
* Detergents
* Heat
* pH
* Protein elutes off by binding free ligand
* Binding may not have been reversed so free ligand competes for active site
Size-Exclusion Chromatography
- Relies on porous beads
- Larger molecules elute first because not trapped in small pores
- Smaller particles trapped retaining in column
Protein Isolation
- First step in analysis
- Protein identity must be confirmed by amino acid analysis/activity
- With unknown proteins, want classification of feautures
Activity Assay
Display lower actvity after concentration determination:
1. Contamination with detergent
2. SDS could yield artifically increased protein level leading to lower activity than expected (calculated higher than actual)
3. Enzyme denatured during isolation and analysis
Edman Degradation
Required sequential degradation for amino acid sequencing
* Proceeds from amino (N-) terminus
Electrophoresis
Uses gel matrix to observe migration of proteins in response to an electric field
* Can only handle small volume of protein
* When isolating protein, want a pH that makes protein A negative while B and C are neutral/positive
Antibodies
- Specific to single antigen
- Each B-cell prduces single type of antibody with constant region specific to host and variable region
- Label antigens for targeting by other immune cells
- Can cause aggltination by interaction with antigen
- Have two heavy chains and two light chains
Trimeric G Proteins
Have a, B, and y subunits in ALL
a Subunits:
Gs, Gi, Gq (differ depending on G protein-coupled receptor’s function)
pI
Determined by relative number of acidic and basic amino acids
Basic Amino Acids:
Arginine, lysine, histidine
Acidic Amino Acids:
Aspartic acid, glutamic acid
Glycine is least contributor of all amino acids
