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BCH 451 - Exam 1
Terms in this set (207)
-Tendency for these hydrophobic molecules to coalesce or group together
-Process is entropy driven
Why the hydrophobic effect?
Ordering of polar H2O molecules around non-polar molecules minimized when multiple non-polarmolecules group together
Hydrogen Bond Definition
Weak attraction between a slightly positive hydrogen atom and a slightly negative atom
What happens to H-bonding when you heat H2O?
There are less hydrogen bonds
Critical Features for H-bond Strength
1) Defined Length
pH equation (with hydrogen ions)
pH=pKa+ log(con. base/con. acid)
At the midpoint of the titration curve....
There is an equal ratio of conjugate acid and conjugate base
Regarding the Henderson Hasselbach equation, when pH is equal to pKa...
The ratio of Con.Base and Con. Acid are equal
If the pH given is less than the pKa on the table
Add a proton
If the pH given is less than the pKa on the table...
Make it an ion
Carbon-Carbon bonds are....
Functional groups make aliphatic molecules
Electronegative and Polar
-increase functional diversity of the molecule
-create a polyfunctional molecule
Functional groups provide sites for
-Ionic interaction (charge-charge)
The heat and energy transformations of biomolecules and ch
Any energy is retained within the system during any exchange or transformation
Energy is exchanged between the system and the surroundings
3 Things Chemical Reactions are controlled by
First Law of thermodynamics
Energy cannot be created nor destoryed
Delta H of a rxn
Second Law of Thermodynamics
Systems/Molecules tend to be more random/disordered
Nucleic Acid Monomer
Biological Macromolecules are....
3D, flexible, dynamic
Protein Functions (8)
2) Store and transporting other molecules
3) Serve as membrane channels for cell transport
4) Serve as structural components of cells, organelles, and tissues
5) Mechanical motors for movement of cells and cellular components
6) Regulators of gene expression in replication, transcription, translation
7) Serve as receptors for cell signaling and intracellular communication
8) Specialized functions such as antibodies and hormones
Important structural feature in amino acids...
The carbon that is connected to the side chain is chiral
All organism have D or L type of proteins
-The average on the titration curve of two pKas
-Zero net charge
-Linear sequence of amino acids in a short peptide
-Established by the peptide bond resonance b/c no free rotation
Local conformations of folded proteins maintained by H-bonding of amino acids within the protein
-Folding of the secondary structure to get a 3D shape
-Major force would be the hydrophobic forces in the interior and hydrophillic interactions with the solven
The association of two or more folded proteins into a multi-subunit complex
Forces used to fold and stabilize a protein
2) Charge-charge interactions
3) Van der Waals
4) Hydrophobic Interactions
Relative Strength of Biomolecule Weak Interactions
Electrostatic > H bond > Hydrophobic> Van der Waals
Weak interactions are used for...
The structure, folding , and interaction of all biological molecules
Angle around the N-Carbon(alpha)
Angle around the Alpha carbon-Carbonyl carbon bond
Atoms sharing outer shell electrons to fill the other shells
Transfer of electrons from one atom to the other creating charged ions
Covalent Bond Strength
Difference in atom electronegativities
Draw a phopsphate ester
Draw a phosphoanhydride
Draw a phosphodiester
The equilibrium constant defines ratio of products to ratio of reactants
When a reaction is not at equilibrium
The reaction is driven to be moved towards equilibrium
Magintude of the force driving a reaction....also called change in free energy
Standard State of Energy
1ATM, [1M] concentration of reactant and product, 25 degrees C, pH=0
Gprime equation equals...at equil
How would you increase the rate of more product?
-Add more heat
-Add more reactant
-Lower activation energy by adding an enzyme
The free energy of a biological reaction ...
The summation of enthalpy and entropy
Draw proline in the middle of a chain
Physiological Salt [NaCl]
Van de Waals Interaction
-Dipoles influencing other dipoles
-Electron cloud around an uncharged atom influences the neighboring "uncharged" electron cloud.
-Interacting dipoles exhibit a slight attactive force between atoms b/c the attraction/repulsion strength is distance dependent
Strength of Van de Waals Interactions
-Specific angles show how specific amino acids promote the forming of secondary structure using those amino acids
Rotation of the peptide chain at the Alpha carbon allows
Peptide chain to be flexable allowing the formation of different secondary structure
Type of secondary structure
Alpha helix and beta sheets
-Right handed helix is formed by interaction of H-bonding which is an H-bond every 4th amino acid
-The side groups are pointed outward, and aren't involved in Hbonding but can stabilize the structure
-Right handed helix makes a turn once every 3.6 AA residures
-Preferred by small uncharged molecules
What type of side chains are preferred in an alpha helix?
An hydrogen bond contains how many atoms? Backbone atoms?
The right-handed alpha helix makes one turn....
Every 3.6 residues
Are the side groups of the amino acids in alpha helicies involved in H-bonding? What is their purpose?
No they are not. But they help stablize their structure
-Inter-strand hydrogen bonding of beta strands to form beta sheets
-A beta strand in a peptide chain is almost fully extended
R groups in beta sheets are...
Trans above and below the strand
H Bonding in Antiparallel beta sheets
H bonding in Parallel beta sheets
H bonding in beta strands occurs between...
The carbonyl oxygen and amide hydrogen
Loops and Turns
-Connect alpha helicies and beta strands, but may be up to 10% of the totall protein length
-Change directions of peptide chain
-Glycine does not apply since it's so small
-Connect antiparallel beta strands
-Smaller loops of about 4 residues
Folded tertiary structures can be stabilized by..
Disulfide bonds...they are made by oxidizing Cys groups to make a S-S bond.
-They bring polypeptide chains together
Combinations of alpha helicies and beta strands forming distinctive structures that are found in different proteins
-Large, independently folded compact regions of a protein structure within a folded protein
-Structural domains are typically functional
-DNA Pol 1 is a good example
Polymerase enzymes are as....
-Open hand which is conserved in all polymerases because it was conserved in all organisms
Forces between individual protein subunits
-Primarilly hydrophobic forces
Why are weak interactions important for the subunits?
-Important for complex function because you can associate and dissociate easily which is important for regulation
Who knows about protein folding?
Protein Folding Steps
Active enzyme ---protein denautration ---> Inactive enzyme ---protein renaturation---> Active Enzyme
What drives the process of protein folding?
But most importantly, the hydrophobic effect
Protein chain unfolded with covalent bonds broken
Protein chain is destroyed with colvanet bonds cleaved
-The melting temperature of a protein that is trying to be denatured. Typically at the 50% mark.
-Protein denaturation is a cooperative process
Why is protein denaturation a cooperative process?
Because it unfolds over a narrow range of temperatures
Two times of proteins
Fibrous and Globular
Fibrous Proteins Description
1) Long chains
2) Hydrophobic amino acids
3) Not soluble in H2O
4) High tensile strength
5) Secondary structural features of the protein dominate
Globular Protein Description
1) Spherical 3D structure
2) Charged polar amino acids on the sruface
3) Hydrophobic amino acids in the interior
4) Soluble proteins
-Alpha keratin helix is a right handed helix
-2 right handed helices in a coiled-coil make a left-handed helix
-Coiled coil helices make a protofilament (2x2)
-Protofilaments wrapped in a bundle make a filament
A key feature of alpha keratin
-Disulfide bond formation between helical chains
-Insoluble protein, so the predominating amino acids should be hydrophobic
-The more disulfide bonds crosslinking...the harder the alpha keratin
-Triple Helical Cable
-3 left handed helices forming a right-handed superhelix
-Amino acid Gly-X-Y with x and y often being proline
How does the triplet repeat of Gly-Pro-Pro elp stabilize the structure of the 3-stranded RH helix?
-Because it's stabilized by the steric repulsion of the Proline rings located on the outside of the helix.
-Since the Glycines are in the 3rd position of the chain, they are located on the interior of the 3-strand helix since it's the only one that can fit
-Antiparallel beta strands stabilized by Hydrogen bonding
-Turn into beta pleated sheets
-Amino acid sequence is Gly-Ser-Glu-Ala-Gly-Ala
Why if the repeating 6 Amino Acid Sequence Important for fibronin Structure?
Allow beta sheets to stack up and therefore interlock
Beta sheets are stabilized by...
Beat pleasted sheets are stabilized by...
Van der Waals Forces
Proteins of the globin family
Hemoglobin and myoglobin
Stores and delivers O2 to the muscle
Transports oxygen to the blood
How do proteins bind to oxygen?
They use a prosthetic group which in this case is heme.
What is heme composed of?
-Heme group is a planar structure
-Binds to the hydrophobic pocket of myoglobin/hemoglobin proteins
-There is no H2O in the site, the Fe2+ remains Fe2+ and does not become oxidized
Main forces holding tertiary structures together...
-Forms stacked beta pleated sheets that are antiparallel
-Amino acid sequence is Gly-Ser-Gly-Ala-Gly-Ala
Why is the repeating 6AA sequence important for its structure?
The alternating Gly & Ala on the opposite side allow the stacking of Beta sheets thus having interlocking happen
Beta Pleated Sheets are stabilized by...
Van der Waals forces
Beta Sheets are stabilized by..
Fibrous secondary structure vs. Globular Secondary structure
Fiberous: Composed of one Secondary structure
Globular: Composed of several secondary structures
Globular Protein Folding
Gloular protein structural diversity
Reflects functional diversity
Are globular proteins rigid or nonrigid?
Globular protein surface
Interacts with other molecules
How do proteins bind oxygen?
1) Nitrogen of each pyrole ring coordinates the binding of the Fe2+ atom
2) 4 Pyrole rings are covalently linked by methyl groups
3) Heme group from a plantar structure
4) The heme group binds in hydrophobic pocket of myoglobin/hemoglobin proteins
5) No H2O, so Fe2+ remains ferrous
-Important because of the aspect of its function
-7alpha helices/chain --> 2 alpha chains
-8alpha helices/chain--> 2 beta chains
-Myoglobin and hemoglobin are in the same family
-Exhibit similar folded structure
Why do members of a protein family exhibit similar structures?
-They are all evolutionarily related from a common ancestor
Myoglobin and Hemoglobin bind_________ to O2
Un bound O2
The extend of O2 binding...
-Depends on the concentration of O2
-Measured as partial pressure (pO2 in units of torr)
Saturation curve of myoglobin
-Myoglobin has a single binding site and a high affinity for oxygen, making it quickly saturate
Saturation curve of Hemoglobin
-Hemoglobin has a tetramer for O2 binding sites
-Affinity for O2 is initially weak, but strengthens as more binding occurs
Why does globin-heme affinity for oxygen increase
-As O2 binds, the structure of the global tetramer is altered, and the binding affinity for O2 binding increases
-this is called...allosteric regulation
Hemoglobin is a...
Oxygen is a...
Binding of the first effector molecule increases the binding affinity for the next effector molecule
The binding of the first effector molecule decreases the binding affinity for the next molecule
Oxygen binding by hemoglobin is regulated by..
2) Carbon Dioxide
3) H+ ions
-Binds a central cavity in the hemoglobin tetramer
-Confers cooperative O2 binding on the hemoglobin tetramer
-Affects O2 affinity
The presence of BPG in red blood cells...
Allowsthe binding of successive oxygens to alter the binding affinity of additional oxygens
-Only 1 binds the hemoglobin tetramer
-Stablized by interactions of BPG negative charges with Lys and His
Carbon Dioxide and H+
-A product from respiration and is transported to the lungs for expulsion
-Remaining CO2 is carried by hemoglobin
Most transported in blood as bicarbonate
CO2 + H2O --carbonic anhydrase---> HCO3_ + H+
The Bohr Effect
-Unionized amino groups of hemoglobin can react reversibly with CO2
-Hemoglobin-NH2+CO2 ---> HemoglobinHugeComplex + H+
-CO2 + H2O --carbonic anhydrase---> HCO3_ + H+
-pH is lowered which causes AA Side groups to be protonated which stabilizes the deoxy form of hemoglobin
-Two His residues stabilize heme binding
-If any mutate to Try, the Fe2+ in heme is now Fe3+ which means O2 cannot bind to heme.
-Now, hemoglobin can only bind to 2 oxygens, causing HBM
Blue or chocolate brown skin
Sickle Cell Anemia
Valine 6 goes to Glumatic Acid 6
-Hydrophobilic uncharged -> Hydrophilic Negative Charge
-Protects against malaria but causes blood cell collapse
-Hemoglobin tetramer is very sticker and has a bunch of interacting pairs of hemoglobin molecules
-The deoxy form of hemoglobin promotes fiber formation
-As hemoglobin gives up O2 in the tissues, fibril formation promotes cell sickling in tissues and this tends to block
Alleosteric Effect (in regards to the sigmoidal curve)
-As oxygen binds, the structure of the global tetramer is changed, leading to a conformational change, increasing the affinity for O2, explaining the sigmoidal curve.
Strategies for Isolating/Purifying a Protein
A) Select a protein source
B) Solubilze the tissue or cell
C) Protein fractionation/Purification
D) Protein analysis/chracterization
Selection of protein Source
-Find protein abundance in the selected tissues or cells
-Abundance/availlability of selected tissues/cells
-Ability to work with selected tissue and cells
-Concentration of protein in a defined cellular organelle or compartment
Strategy for Isolating/Purifying a Protein
A) Selection of a protein source
B) Solubilization of tissues and cells
C) Protein fractionation
D) Protein analysis
Selection of tissues/cell source for protein purification
1) Protein Abundance
3) Ability to work with tissues
4) Concentration of proteins in a defined cellular organelle
Consideration during Purification
-Be sure to maintain the native conformation of the protein.
-Be gentle and make sure physiological conditions are maintained like pH, ionic concentration, temp, and mechanical agitation
5 things to be careful for when isolating a protein
2)physiological ionic strength
3) low isolation temperature
4) Maintain sub cellular integrity
5) Minimizing Mechanical agitation
Maintaining physiological pH
-Important due to interactions especially with the polar solvent.
-Use weak acids to buffer such as Tris, -HCl, and phosphate buffers
Maintaining Physiological ionic strength
-Critical for maintaining the salt bridges for protein folding as well as the correct ionic strength/polarity
-Metal ions can act in protein folding
Maintaining Low Isolation Temperature
-Proteins are easily denatured and can be destroyed by high temperatures.
-Important to keep the temperature at around 4 degrees Celsius, usually used to keeping things on ice.
Maintaining Subcellular/Organelle Integrity
-Remove harmful degradative enzymes like proteases. Prevent the harmful degradative enzymes such as proteases.
Minimizing Mechanical Agitation
-Proteins are easily denatured by solution agitation.
Methods of Breaking open tissues
Has spinning blades that tear up tissues and breaks open cells
-Shearing force disrupts tissues and breaks open cells.
-Up and down movement of ball within the vertical chamber forces tissues open and breaks open cells
Methods of Breaking Open Single Cells
1) Mechanical shearing
3) Osmotic lysis
4) Detergent membrane solubilization
5) Enzyme digestion
-Disrupt cell membrane but leaves internal organelles intact.
-Prevents leakage of organeller components
-Metal rod is immersed in a suspended cell solution and the probe is ultrasonically vibrated. ---Strong vibrations disrupt the cell and destroy only the membrane, keeping the other organelles intact
-Cells collected by centrifugation are put in a hypotonic buffer and water rushes in and busts the cell membrane
Detergent Membrane Solubilzation
-Nonionic detergents are used to soluble cell membranes
-Detergent inserts itself into the cell membrane, causing disruption
-Introduce an enzyme that consumes the cell wall such as lysozyme or zymolase
-Centrifuge tube with tissue cells and put under a centrigifal source.
-Lighter and slower are higher & heaver faster are lower
Protein Fractionation Based Upon
1) Protein Polarity - differential solubility
2) Protein Size - sedimentation gradient centrifugation, size exclusion chromatography
3) Protein Charge- ion exchange chromatography
4) Protein Affinity - affinity chromatography
Protein Analysis Techniques
1) Primary Sequence - Edman degradation sequencing, mass spectrometry sequencing
2) Molecular Weight - SDS gel electrophoresis
3) Structure - X ray crystallography
Protein Fractionation/Isolation is based on.....?
Protein polarity and protein solubility
The differential precipitations of proteins from solution at different increasing concentrations of salt
Increasing the salt concentration of the solvent...
Alters the solvent (H2O) character
The more water molecules now hydrating the salt ions...
The few water molecules hydrating salt ions, and the proteins become dehydrated
Salvation or hydration of the protein is altered, and protein dehydration leads to a bunch of proteins
Different proteins are solvated or hydrated differently at elevated salt concentrations so.....
Different proteins will be precipitated at different salt concentrations
Most commonly used salt
-At different concetrations of NH4SO4 different sets of proteins precipitate, with 3-4 gold enrichment.
-PPT collected by cfugation and is resuspended in the buffer which is used to obtain a final salt
Protein Fractionation is based upon ....
Sedimentation rate of a protein through a sucrose or glycerol gradient is based upon the protein size
In a centrifugal tube, where does the gradient increase?
Going down the tube
Gel Filtration Chromatography
Separates proteins based on molecular size. The proud beads allow proteins that are smaller to filter through while the larger proteins simple fall through faster.
Proteins that are excluded from entering the beads because they're too big
Ion Exchange Chromatography
-Has beads that carry a positive or negative charge and are carried over a protein matrix.
Anion Exchange Chromatography
Has a positive charge that binds to negative proteins
Cation Exchange Chromatography
Has a negative charge that binds to positively charged proteins
Process of ion exchange chromatography
1) All the positively or negative charged proteins first bound to the column
2) Proteins bound at low salt and unbound washed
3) Bound proteins then serially eluted with increasing salt
The same things as ion exchange , but the matrix beat has a covalently attached ligand it should also be done at conditions that preserve the structural integrity
Potential Ligands for Affinity Chromatography
Substrate analog, interacting protein, coenzyme/cofactor, antibody
Potential buffer eluates for Affinity Chromatography
High salt. Altered pH. Excess free ligand. Elevated Temp.
How do you detect a certain protein in a sea of proteins?
-You must consider specificity and sensitivity.
-Cannot do size, but you can do binding affinity, antibody recognition, and enzyme activity.
1.0 unit of enzyme activity
-1.0micromol of substrate catalyzed to product under optimum conditions at 25 degrees C
number of enzyme units permg protein
% of total starting protein /enzyme in final purified protein fraction
Edman Degradation Procedure
-Tags the N terminal end of a peptide
-The N-terminal tagged amino acid side is cleaved
-Identity of tagged amino acid is determinded by chromotography
-Repeat the procedure with remaining peptide with one amino acid shorter
-Chemically tags the N terminal amino acid
Mass Spec. Sequencing
-Measured the mass to charge ratio of ions that allows derivation of molecular weight
Mass Spec Steps
1) Charged peptides are released in a mass spectrometer
2) Ions are accelerated by a high voltage e-field
3) Detecter measures time of flight which is proportional to the molecular weight of the peptide, which is very precise
Draw Mass Spec Curve
SDS Polyacrylamidde Gel Electrophoresis
-Individual Protein samples are loaded onto separate lane wells and then an electric field is applied.
-Negatively charged proteins migrate from the well to the gel matrix towards the cathode.
-Smaller proteins are able to migrate faster through the dense matrix and the larger ones stay put.
-Rate of protein migration through this gel matrix is dependent upon the weight
-Disrupts and denatures protein structure
-Sodium dodecyl sulfide - CH3(ch2)10CH2OSO3- Na+
-Hydrophobic tail disrupts hydrophobic interactions
SDS binding to protein
-Denatures and linearizes protein (look at notes)
-Look at notes
SDS -protein overall charge
-Charge proportional to protein mass
Determination of protein molecular weight
-Can be determined by the presence of weight markers.
Gel electrophoresis other uses
-Can be used to monitor the purity of a protein during isolation protocol
X ray crystalography
-Determines the detail of secondary and teritary structure
-Sophisticated and precise
Xray crystalography process
X ray beam -> goes through sample thats inside the crystal -> creates scattered radiation and goes through a detecter-> then angle of scatter and distance of spacing spot intensity
Angle of Scatter and Distance
-Mathematically solves for the structure of the molecule
-And is crystal specific for the strcutre of molecule
Crystal structures of molecules are described in terms of....
"Resolution" which is the ability to distribute
-The smaller number of A the able, and yo have the ability to resolve atoms in the molecule that are separated by the indicated number of angstroms