Cell Structure and Function: Part 1
1. Describe the basic cellular architecture and specialized functions of the eukaryotic cells.
o Plasma Membrane – contains cholesterol (~50%, promotes membrane stability), phospholipids
(~50%), sphingolipids, glycolipids, and proteins. Only the noncytoplasmic side of membrane
contains glycosylated lipids or proteins (i.e. the plasma membrane is an asymmetric, fluid
bilayer).
o Cytosol – Unstructured Aqueous Space
o Mitochondria – double membrane organelle, which contains its own DNA (~16,569bp, circular
and present in multiple copies; Matrilineal) and is responsible for oxidative phosphorylation
(acetyl-CoA production, Krebs cycle, Fatty acid oxidation β-oxidation).

On the inner membrane cristae, globular membrane associate protein units are
responsible for producing ATP.

Also, Mitochondria are responsible for Detoxification.

Contains inter-membrane space and matrix space (inter-cristae space

Matrix contains mitochondrial DNA and enzymes.
o Nucleus – Membrane bound organelle, which contains DNA in the form of Chromosomes. RNA
synthesis and processing, along with ribosome assembly occur here.
o Nucleolus – Large structure in nucleus where rRNA synthesis and processing, and ribosome units
are assembled.
o Nuclear Envelope – Double-membrane surrounding the nucleus. The outer membrane is the ER.
Also, separates the chromatin from the cytosol.
o Endoplasmic Reticulum (ER) – network of membrane structure. Largest Membrane System.

RER  w/Ribosomes on outer surface  Protein Synthesis, segregation and processing
of secretory and membrane proteins with posttranslational modifications. Associated with
the nuclear envelope.

SER  wo/Ribosomes  Triglyceride, Cholesterol and Steroid Synthesis. Detoxification
of Drugs.
o Golgi Complex/Apparatus – Proteins and lipids synthesized and packaged in the RER are
transported to the Golgi for post-translational terminal modification (glycosylation) and final
packaging. This complex consists of a series of flattened, slightly curved membrane-bound
cisternae.

Cis-face (closest to rough ER; convex shape, the entry phase)

Medial face

Trans-face (concave, the exit face)
o Centrioles – microtubule polymerization center.
o Vesicle – Small membrane bound organelle that can be used for storage or subsequent secretion.
o Peroxisome – Small organelle involved in long fatty acid chain and amino acid degradation via
hydrogen peroxide Oxidation.

Contains 40-50 Oxidative Enzymes: Urate Oxidase, and Catalase e.g.

2 HOOH –Catalase 2H2O + O2
o Lysosome – Small degratory organelle w/pH 4-5

~40 different acid hydrolases: Sulfatases, proteases, nucleases, etc. Which have been
targeted by M6P and its receptor (recycled).

Actively pumps H+ into lumen via ATP-dependent H+ pump.

Early Endosome via Clathrin dependent pathway has yet to fuse with the Lysosome and is
pH 7.4.

Late Endosome has fused with lysosome and is ~pH 5.0

Lysosomes are also involved in Phagocytosis and Autophagy, which is Programmed
Organelle Death (e.g. Mitochondrion).

Residual Bodies contain the degradation products and are eventually eliminated from the
cell (exocytosed).
o Cytoplasmic Inclusions – Dense looking bodies:


Glycogen – storage form of glucose found in live and muscle cells.

Lipid Droplets – Triacylglycerols stored in a membrane bound droplet.

Pigments – Possess color in its natural state (e.g. hemoglobin, melanin, lipofuscin).

Crystals – Crystal of Charcot Bottcher in Sertoli Cells of the testis and some interstitial
cells.
KEY POINTS IN ORGANELLES:
o Proteins synthesized in RER are moved to Golgi for sorting, packaging and trafficking.
o Steroids, lipids are synthesized in the SER. Detox occurs here as well.
o Endosomes internalize plasma-membrane and proteins along with soluble materials from the
outside, sort back to membrane or to lysosomes.
o Mitochondria have a highly permeable outer membrane and protein rich inner membrane which is
extremely folded (cristae). Enzymes in mitochondrial membrane and matrix carry out oxidation
of lipids and sugar coupled to ATP synthesis.
o Peroxisomes oxidize organic compounds without production of ATP. Catalase reduces amount of
left over HOOH.
o Lysosomes have acidic interior, contain many hydrolases, which degrade materials.

Functions:
o Lipid Droplets – storgage.
o Movement – Phagocytes
o Contractility – Muscle Cell
o Conductivity – Nerve Cell
o Enzymatic Synthesis & Secretion – Pancreatic Acinar Cells
o Mucous “ “ – Mucous Gland Cells
o Steroid “ “ – Adrenal Gland Cells, testis and ovaries
o Ion Transport – Kidney Cells
o Intracellular Digestion – Macrophages and some types of WBCs.
o Physical & Chemical Transducers – Sensory Cells
o Metabolite Absorption – Intestine, and kidney.
o Motility, contractility, conductivity, absorption, synthesis (assimilation), respiration, secretion,
excretion, growth, reproduction, and death.
2. Explain the unit of measurements related to the study of the cells.
o 1in = 2.54cm
o Human eye limit 100μm
o Cells – μm-cm
o Organelles – nm-μm
o Macromolecules (proteins) 50-500nm
o Small Molecules (amino acids) 1nm or ~10A
3. Compare the basic techniques to study the cells and subcellular organelles.

Light Microscopy
o Maximum Resolving Power = .22μm

Magnification is from 40 – 1500X
o Tissue Preparation:


Embed Tissue Sample in Parafin

Add fixative and dehydrate

Sectioning (4-5μm)

Staining

Observation

Photomicrograph
Electron Microscopy
o Theoretical Resolution = .1nm  Actual Max = 1-2nm

Magnification is from 1000s –400,000sX
o Tissue Preparation:

Embed Tissue Sample w/in Plastic

Fix sample with osmium tetroxide

Thin Sectioning (~100nm)

Staining with Heavy Metal (Au, Hg etc.)

Observation

Electron Micrograph

Interpret Planes of Section: Refer to Sectioning Quiz.

Isolation of Cell Constituents: Refer to Differential Centrifugation Quiz

Culturing of Animal Cells: Refer to Cell Culturing Quiz
4. Review the structure and function of plasma membrane.

Plasma Membrane Composition:
o ~Phospholipids:Protein (1:1) on average
o Glycoproteins with Carbohydrate moieties
o Glycolipids
o Cholesterol
o Outer Leaflet with E face
o Inner Leaflet with P face

Proteins Act As:
o Receptors, recognition, binding units, and anchors.
o Pores or channels: moving ions or electrolytes (Na+, K+, HCO3-)
o Enzymes: drive active pumps to promote K+ concentrations inside the cell.

Plasma Membrane Functions As:
o Structural Integrity
o Interfacing unit between the cytoplasm and the external environment
o Regulate the movement of substances
o Cell-cell communication, interaction and recognition.

KEY POINTS REGARDING MEMBRANES:
o Membrane system includes all of the plasma membrane and membrane-bound internal
compartments (organelles, vesicles)
o Internal membrane surface far exceeds Plasma membrane surface area
o Phospholipid bi-layer is a sheet with hydrophilic faces and a hydrophobic core impermeable
to water soluble molecules and ions.
o Most lipids and proteins are laterally mobile in the bi-layer.
o Define the difference between Integral and Peripheral proteins
o Integral proteins may be transmembrane spanning.
o The nucleus and mitochondria have a double membrane with all other organelles having one.
5. Explain the membrane transport and the process of phagocytosis and pinocytosis.
o Phagocytosis – Cell eating, and takes the diameter of >250nm. Usually receptor mediated.
o Pinocytosis – Cell drinking, and takes the diameter of <150nm. Also responsible for membrane
recycling. Sometimes receptor mediated.
6. Illustrate seven intracellular organelles and match them with their function(s).
o Refer to Objective 1 for the answer.
7. Compare four types of abnormal functions of cellular organelles and two types of abnormal functions
of the cells.
o Tay-Sachs disease – which is marked by the enzyme deficiency of Hexosaminidase A in the
lysosomal compartment, which is responsible for breaking down certain glycolipids, which
results in the build up of gangliosides and eventual cell death due to over storage in brain tissue.
o I-cell disease – is the result of a broken GlcNAc Phosphotransferase enzyme, which is responsible
for altering the mannose glycosylation in the Golgi Complex to its M6P form. The M6P is
recognized by the M6P receptor and is used to target acid hydrolases to the Lysosomal
compartment. Without this targeting, the hydrolases are expelled to outside of the cell. The
result is inclusion bodies in fibroblasts that do not get broken down, and just build up over time,
causing cell death.
o Kartagener’s syndrome – lack of dynein in cilia and flagella. This results in lack of arms
extension from the doublet fused microtubules. Thus rendering the cilia and flagella immotile,
resulting in male sterility and chronic respiratory infection.
o Pro-insulin diabetes – Defect of the Pro-insulin cleaving enzyme in the secretory granule. No
morphological change is known. Result is High blood pro-insulin content with diabetes.
o Hypoxic Vacuolization caused by viral infection.
o Storage of Metabolite Abnormalities:

Lysosomal storage disease

Glycogen storage disease

Lipid storage disease

Pigmented material storage disease
o KEY POINTS FOR ABNORMALITIES:

Cell can respond to physiological/pathological stimuli by changing their functions.

These changes are reflected in altered cellular morphology

Vacuolization is a common response to physiological and pathological stimuli

Storage of metabolites occurs during metabolic disorders

Nuclear changes such as clumping, chromatin condensation along the nuclear membrane,
etc, characterizes cellular death

Cell membrane rupture or injury (necrosis) represents this as well

Apoptosis is required as a normal cellular function.
Cell Structure and Function: Part 2
1. Compare the structure and functions of microtubules, microfilaments and intermediate filaments.
o
Microfilaments/Actin –




Structure α, β, & γ–

Thinnest filament at ~6nm

G-actin (globular) monomers make up the F-actin (filamentous [2-stranded helix])
polymer via a nucleation process that is polar.

There is a (+) end, rapidly growing, and a (-), slow growing end (actually
dissociates).

Ca2+, ATP and G-Actin are needed for assembly at the (+) end
Actin Cross Linking/Binding Proteins-Bundles/Parallel:

Fimbrin & α-actinin resides in the core of the epithelial brush border microvilli;
provides membrane adhesion of actin (adhesion plaques).

Fascin & villin cross-links parallel or bundled actin in regular arrays.
Actin Cross Linking/Binding Proteins-Network:

Spectrin resides in Erythrocytes and binds actin network to plasma membrane
through Ankyrin and Band 3 dimer. Disease of Hereditary Spherocytosis.

Filamin resides in Platelets and binds actin network to plasma membrane through
Gp1b-IX transmembrane protein. Help maintain cellular shape/cortex.

Dystrophin resides in Muscle cells and binds actin network to plasma membrane
through a Glycoprotein complex. Disease of Muscular Dystrophy.
Actin Cross Linking/Binding Proteins-Contractile:




o
Myosin resides in Muscle tissue and provides framework for the Sliding filament
model of muscle contraction. Also responsible for the contractile cleavage
furrows.
Actin Polymerization Control:

Cofilin aids in (-) dissociation

Severin and Gelsolin Severing and (+) capping for turn over

CapZ (Cap (+))

Tropomodulin and Arp2/3 Cap (-)
Action of Key Drugs on Actin Polymerization:

Cytochalasins – Secreted by various fungi. Binds and caps the (+) end to prevent
further polymerization. Prevent Cell Division.

Phalloidin – Another fungi toxin. Binds and stabilizes F-actin from
depolymerization. Essentially kills and destroys the cell.
Key Points for Actin:

Actin Cytoskeleton is dynamic

(-) and (+) growth rate asymmetry

Capping and Severing proteins for control, length and stability

Regulating polymerization can create movement or shape

Crosslinking proteins in bundles and networks

Microvilli fimbrin and villin
Intermediate Filaments –


Structure – Parallel dimer, combines with another parallel dimer in an antiparallel fashion
to form an antiparallel tetramer. This antiparallel tetramer is the beginnings of a
protofilament. Two Protofilaments combine in a parallel fashion to form a protofibril.
Four Protofibrils form an Intermediate Filament.

2 Dimers combine Antiparallel  Antiparallel Tetramers (protofilament)

2 Antiparallel Tetramers (protofilaments) combine parallel  Protofibril

4 Protofibrils combine  1 intermediate filament.

1 IF = 4 Protofibrils = 8 Protofilaments = 16 Dimers = 32 Monomers
Principle Types of Intermediate Filaments:

Nuclear –
o Type V are Nuclear Lamins A, B, and C, which reside in the Nucleus.


They control the assembly of the Nuclear Envelope and
organization of perinuclear chromatin.

A –Binds (Bmembrane bound)Binds—C
Cytoplasmic –
o Type I & II are Acidic and Basic Keratins present in the Cytoplasm of
Epithelial cells.
o Type III are

Vimentin – Mesenchymal tissue (fibroblasts)

Desmin – Muscle tissue

Glial fibrillary acidic protein – Glial cells, astrocytes

Peripherin – Peripheral and Central neurons
o Type IV are NeuroFilaments- L, M & H, which are present in Mature
Neurons.

o

Provides 3-D structure for cellular organization, which is adaptable for
cytoplasmic/membrane (plasma as well as nuclear envelope) rearrangements.

IF are made up of many different types of monomers, which are tissue specific.

IF are very strong and provide tensile strength. IF crosslink plasma membrane,
nuclear membrane, microtubules and microfilaments.

These filaments do not polarity like microtubules or microfilaments.
Microtubules –





o
Key Points for Intermediate Filaments:
Structure – Microtubules are made of α and β tubulin dimer subunits.

These dimer subunits polymerize with the help of GTP (GTP driven Growth),
Mg2+ and other MAPs (microtubule associated proteins), to make a protofilament
assembly

Protofilament assembly  Sheet assembly  and finally Microtubule Elongation
(13 protofilaments). 25nm. (+) end is fast growing and (-) end is slow growing.
Types of Microtubules A, B, C (C fused to B fused to full A)–

Singlet – in the center of cilia and flagella.

Doublet – periphery of cilia and flagella. Dynein bound for motion.

Triplet – basal bodies and centrioles.
Motor Proteins –

Kinesin  Vesicle (+)

Dynein  Vesicle (-)
Action of drugs –

Colchicine – inhibits monomer addition, thus promoting depolymerization.

Vinblastine/Vincristine – induce crystal aggregates of tubulin.

Taxol – stabilizes polymers, and thus destroys cell by preventing disassembly.
Key points for Microtubules –

MT are Hollow 25nm structures.

They do exhibit structural polarity.

They are made of alpha and beta subunit dimers.

MAPs organize MTs and affect polymer stability
Nucleus

Nuclear Envelope – Lipid bilayer, which contains pore-complexes and is contiguous with
RER and the inner membrane with nuclear lamin.

Nuclear Pore – Composed of three rings Cyto, middle and Nucleoplasmic

o

Cytoplasmic and Middle rings keep pore complex in place

Nucleoplasmic ring has protruding basket

Middle ring contains a transporter complex

Exportin Proteins transport macromolecules >60kDa to the cytoplasm

Importin alpha and beta Proteins transport cargo, i.e. proteins, to nucleus.
Nucleolus –

Fibrillar Center: inactive DNA

Pars Fibrosa: RNA

Pars Granulosa: rRNA synthesis

Chromatin – Heterochromotin is inactive (dark regions), and Euchromatin is
transcriptionally active (light regions).

Centrosome is also known as the MicroTubule Organizing Center. Within the MTOC are
a pair of Centrioles, which direct Microtubule formation. These Microtubules are also
known as Spindle fibers and are involved in Chromosomal separation.

Kinetochore MT attach Chromosomes to MTOC to be pulled apart. Polar MT and Astral
MT are polarized and utilize motor proteins to separate the two MTOCs and thus separate
the two sets of Chromosomes.
Key Points for Lecture:

Nucleus is the information center of the cell, and utilizes gated transport through Nuclear
Pore Complexes, which are also called Nucleoporins.

FG-nucleoporins, line the central transporter channel, and are involved in transporting
macromolecules >60kDa with the help of importins and exportins.

mRNP's are exported by mRNA-exporter.

During mitosis, kinetochore MTs capture chromosomes for separation.

Microtubule assembly and disassembly, with motor proteins are responsible for
chromosomal movement.
Blood: Chakraborty Lecture
1. Categorize the composition of blood and compare the difference between plasma and serum.
o
Blood: plasma, serum and its cellular components is ~6-8% of Tot Body Wt.
o
Average Blood Volume = 5.5L
o
RBC Volume = 2.5L
o
Plasma Volume = 3.0L
o
Plasma = 93% water and 7% other (Albumin, globulins, clotting factors, electrolytes, nutrients:
glucose, a.a., cholesterol, vitamins, waste: urea, creatinine, bilirubin, hormones, etc.)
o
Serum = Plasma – fibrinogen, clotting factors and antibodies.
o
Cells = RBCs, WBCs and Thrombocytes.
2. What are the Cellular Components of Blood?
o
Red Blood Cells (Erythrocytes) –

Biconcave, Reversibly deformable, 90% hemoglobin, 120 life span

[Hemoglobin] Ave.


o
13.6-17.2 g/dl in men

12.0-15.0 g/dl in women
Erythrocyte Count Ave.

4.3-5.9x106/mm3 in men

3.5-5.0x106/mm3 in women
Hematocrit: estimation of packed erythrocytes per unit vol in Blood

40-50% in men

35-45% in women

Spectrin as an important IF under the RBC membrane, which is associated with actin.
Allows RBC to deform when traversing microcapillary domains.

Hereditary Spherocytosis is a disease, wherein ankyrin, spectrin or associated Band
proteins cannot maintain overall cellular biconcave shape. Results in membrane fragility.
White Blood Cells (Leukocytes) –


o

Granulocytes

Neutrophils – Multilobed nucleus, bacteria microphage in connective tissue, contains
X-chromosome Barr Body. Phagocytosis and destruction of bacteria.

Eosinophils – Orange, few in number, bilobed nucleus, allergic response and parasitic
infections. Contains crystalline core and less dense matrix in granules. Phagocytosis
of antigen-antibody complex destruction of parasites.

Basophils – Few in number (<1%), S-shaped nucleus w/large cytoplasmic granules.
Responsible for inflammatory response.
AGranulocytes

Lymphocyte-T – Cell mediated immune response.

Lymphocyte-B – Humorally mediated immune response.

Monocyte – Differentiate into macrophage. Produce cytokines and present
antigen to T-cells.
Platelets (Thromocytes) –

Contain no nucleus. Originate from the cytoplasmic extrusion of giant megakaryocytes in
the bone marrow. Partial nuclear division process.
3. Develop the concept of the development of blood cells.
o
Hematopoiesis = Blood to make.
o
Pluripotential Cells  Progenitor Cells (Multi-Potential; hematopoietic factor determined)
Precursor Cells (Uni-Potential)  Differentiated Cell (Mitotically Incapable)
o
Table of Hematopoietic Growth Factors:
Granulocyte (G-CSF)
Stimulates Granulocyte Formation
Granulocyte & Macrophage (GM-CSF)
Stimulates Gran and Macro Formation
Macrophage (M-CSF)
Stimulates Macrophage Formation
Erythropoietin (EPO)
Stimulates RBC formation
Thrombopoietin
Stimulates Platelet formation from
Megakaryocytes
Stem Cell Factor
Stimulates all types of Blood Cells
Interleukin 3 (IL3)
Stimulates myeloid cell formation
4. Draw a diagram and summarize the structure and function of platelets. See above
5. Compare the different types of white blood cells. See above
6. Compare the basic concept of the difference between humoral and cell-mediated immunity.
o
Humoral immunity – Activated B-Lymphocytes produce Plasma Cells which produce antibodies,
which circulate throughout the body. IgG & IgM  Immunity against bacteria and viruses. BCells do not interact with MHC receptor.
o
Cell-mediated immunity – Produced in Bone Marrow and migrate to the Thymus. Presented with
antigen by lymphocytes, macrophages, etc. using a MHC protein. T-Helper interacts with Class
II found on Macrophages and B-Cells. T-Cytotoxic Cells interact with Class I found on most
nucleated cells. T-Helper Cells divide into Cytotoxic and memory cells.
7. Abnormal WBC: T-cell destruction in HIV  CD4 T-Helper Cell  viral multiply  Cytolysed.
“Suppressed Immune Response”
Introduction to Proteins: Part 1
1. Describe the most common types of chemical bonds in biochemical systems.
o Covalent bonds – sharing electron pair of atoms
o Electrostatic interactions – depends on electrical charge of atoms.

E=kq1q2/Dr

E = Energy of dissociation

K = proportionality constant 1,389 kJ/mol

q1 and q2 are electrical charges on the atom = 1 in most cases

D = dielectric constant (intervening medium) that opposes electrostatic interactions between
q1 and q2; water D=80

R = distance of the two atoms in angstroms
o Hydrogen Bonding – A weak version of an electrostatic interaction. Contains a dipole moment.
Equal and opposite forces, but uneven.
o Van Der Waals Interactions – More Specifically London Dispersion interactions dictate the
‘instantaneous’ dipole moment within a given set of atoms. Also, there is a ‘contact-distance’
between these non-polar atoms that is dictated by the number of electron shells in the measured
atoms.
o THE NON-COVALENT BOND IS GREATLY AFFECTED BY WATER (H2O).
2. Explain how water can affect chemical bonds.
o Water is a polar molecule with a dipole moment.
o Water is highly cohesive due to hydrogen bonding interactions.
o High dielectric constant D=80.
o Produce hydrated shells, minimizing other electrostatic fields
o Water can thus act as a SOLVENT to REPLACE ELECTROSTATIC INTERACTIONS in
SOLVATED MOLECULES and ATOMS that it comes in CONTACT WITH.
o Total Energy (Kinetic and Potential) remains constant.
o Total Entropy always increases releasing free energy –ΔG in a spontaneous manner. Thus nonpolar and hydrophobic molecules aggregate to release free energy and increasing the overall
Entropy of the water molecules. This also drives the hydrophobic effect, which dictates protein
folding.
3. Define Acid and Base.
o Acid: A chemical compound that donates a H+, or an electron acceptor. A weak acid produces a
strong conjugate base.
o Base: A chemical compound that accepts a H+, or an electron donor. A weak base produces a
strong conjugate acid.
4. Define pH and pK.
o pH is the –log[H+]
o pKa is when a [conjugate acid] = [conjugate base]. Also resistance to pH change is the greatest;
buffering capacity.
5. Explain the Henderson Hasselbalch Equation and its relationship between pH and the ratio of acid to
base.
o Predicts the pH of a buffer, which is determined by –log [HA]/[A-] ratio, rather than –log[H+]
o pH = pKa – log [HAconjugate acid]/[A-conjugate base]
6. Describe the properties of a good biological buffer.
o An acid-base conjugate pair (buffer) resists changes in pH [H+] of a solution.
o A weak acid and strong base make a strong pH buffer.
7. Define a zwitterions and what occurs to it at different pH’s.
o Amino Acids are zwitterions that have electrical charges on amino and carboxyl ends and yet is
electrically neutral. When the pH drop above or below its pI, it’s no longer electrically neutral
because either the amino or carboxyl has become neutral.
8. Write the general structure for amino acids and indicate the alpha carbon.
9. Distinguish between L and D configurations of alpha-amino acids.
o L is in your body. Know the chiral carbon which everything else is linked to.
10. Divide the amino acids into groups based on their side chain characteristics, as well as the 3,1-letter
abbreviations for each.
Introduction to Proteins Part 2:
1. Describe the mechanism of chemical bonding between two amino acids (peptide bond).
o The alpha-carboxyl group of one amino acid is covalently linked to the alpha-amino group of
another amino acid by a peptide (amide or covalent) bond.
2. Define why a polypeptide chain is polar.
o The polypeptide chain is polar because there is an Amino Terminal End (beginning of the
polypeptide chain), and a Carboxyl Terminal End (end of the polypeptide chain).
3. Describe the two primary structural components of a polypeptide chain.
o Backbone – carbonyl group (H-bond acceptor), amine group (H-bond donor; except proline).
Backbones can interact with side chains of other amino acids.
o Side chains (R groups) – variable portion of the protein molecule.
4. Given the mass (kD) of a protein, be able to determine the probable number of amino acids it
contains (or vice versa).
o 1 a.a. = 110 Daltons
-or-
1000 a.a. = 110 kDa
5. Explain the biochemical reversible reaction: cysteine  cystine.
o Disulfide bonds (cystines) are formed by oxidation of the –SH side groups (cysteines).
o –SH side groups are formed by reduction of Disulfide bonds.
6. Distinguish the difference between cis and trans peptide bonds and why the trans configuration is
preferred.
o Almost all peptide bonds are Trans due to steric hindrances from the alpha carbon and R groups.
The one exception is X-Pro; cis.
7. Define what dihedral angles refer to and what restricts them as visualized by a ramachandran
diagram.
H3N—φphi—Cα—ψpsi—COO-
o
+
o
Steric Hindrances dictate the location of the amino acid bonds within the Ramachandran diagram.
8. What are the structural differences between an alpha helix and a beta strand?
o
The α-helix consists of a tightly coiled backbone with its 1) side chains extending outward in a
helical array, 2) α-helices are right handed clockwise screws (righty-tighty), 3) h-bond every 4th
N-H and C=O in the backbone.
o
β-pleated sheet is almost fully extended as a zig-zag type structure, rather than the coiled alphahelix. Adjacent a.a. in the B-strand are 3.5 A apart, in contrast with 1.5 A in the a-helix. B-sheets
can be more structurally diverse than a-helices e.g. Flat and twisted with reverse turns.
9. Explain the differences between a parallel and an antiparallel beta sheet.
o
Parallel – Read frames are in the same direction, and staggered H-bonds between aa’s on opposite
strand.
o
Anti-Parallel – Read frames are in the opposite direction, and the H-bonds between aa’s on the
opposite strand are direct, as opposed to staggered.
10. Describe how reverse beta turns are stabilized in protein secondary structure.
o
The C=O group of residue i hydrogen bonds with N-H group of residue i + 3
Introduction to Proteins: Part 3
1. Within an aqueous environment, what is the expected characteristic of a globular protein’s exterior
amino acid composition, and of it’s interior amino acid composition.
o
Interior of the protein consists mostly of nonpolar hydrophobic residues. And the Exterior of the
protein consists mostly of polar hydrophilic residues. The exception to this rule is when a protein
is embedded in a lipid bilayer or hydrophobic container, which would lend the protein to a
Hydrophobic exterior and a Hydrophilic interior.
2. Define a protein domain.
o
Protein domains are two or more compact globular protein regions connected by a flexible
segment of polypeptide chain.
3. Define a subunit of a quaternary structure.
o
Quaternary structure is the spatial arrangement of proteins containing more than one polypeptide
(or protein) chain (subunits), and the nature of their interactions.
4. What does beta-mercaptoethanol and 8 M urea do to proteins and how can the effects become
reversed?
o
Beta-mercaptoethanol reduces covalent disulfide bonds.
o
8 M urea or 6 M guanidinium chloride disrupts noncovalent bonds.
o
If urea were not dialyzed away, the protein would not re-fold properly leading to randomized
placement of the disulfide bridges.
5. List factors that facilitate protein folding.
o
Cooperative transition (All or Nothing transition) prevents aggregates of ‘mid-folded’ proteins
from developing.
o
Cumulative selection dictates that each bend into the correct shape promotes further correct
folding of the protein.
6. Describe the importance of cooperative transition. See Answer above.
7. How do “knowledge-based” methods provide insights into the 3-dimensional confirmation of
proteins of known sequence?
o
Ab initio (1o sequence prediction) – Determines the differences in various ‘free-energies’ of
different structures as dictated by the 1o a.a. sequence.
o
Knowledge-based (homology based) – Utilizes already known structures of proteins that contain
similar a.a. sequence to predict different structures.
8. Describe the different types of covalent modifications that occur to proteins and subsequent results.
o
Acetyl group – Amino terminal attachment that helps resist degradation.
o
Phosphoserine/tyrosine/threonine – Helps modulate cellular functions.
o
Peptide bond Cleavage – Turns Pro-molecule into active form of protein.
o
Hydroxyl group – Proline, and lysine (Vit C required). Inc. Stability.
o
Glycosylation – Targeting to various IC and EC compartments.
o
Methylation – Also prevents degradation, and changes a.a. contact dynamics.
9. Describe the first two essential steps that must be taken when exploring an unknown cellular protein
for function.
o
Step 1: Purify
o
Step 2: Sequence 1o a.a.
10. Describe when you would choose affinity chromatography rather than ion exchange during protein
purification.
o
Whenever possible. It has the best yield and purification standards.
11. Explain the usefulness of MALDI-TOF.
o
MALDI-TOF – is used to measure the mass/charge ratio of protein fragments, and can be useful
in determining the ‘molecular-fingerprint’ of an unknown protein. These fingerprints are then
matched against a database for identification.
Plasma Membrane: Part 1
1. Describe 3 common types of membrane lipids and discuss amphipathic nature of each. Indicate how
membrane lipid structure facilitates self-assembly of the lipid bilayer.
o
Phospholipids – The phospholipid contains 4 groups. 1) The Hydrophilic Alcohol and Phosphate
group(s). 2) the Glycerol platform/backbone. And 3) the Hydrophobic Fatty Acid 16-18Carbon
tail region.
o
Glycolipids – Meaning Sugar containing lipids. A sugar takes the place of the phosphate.
o
Cholesterol – Based on the steroid nucleus. The majority of the molecule is hydrophobic (4
hydrocarbon linked rings). It has one hydroxyl group, which is hydrophilic, giving it, its
amphipathic properties.
o
Self-assembly of the bilayer is favorable because:

Water exclusion is more energetically favorable

Van der Waals forces from the fatty acid tail regions are energetically favorable

Electrostatic H-bond attractions from the polar head groups are also energetically
favorable
2. Discuss the distinguishing features of integral, peripheral and lipid- anchored membrane proteins.
o
Peripheral – Do not enter or span the hydrophobic core. Removed with mild conditions (salt/pH).
o
Integral – Enter or span the hydrophobic core. Removed only with harsh conditions, such as
detergents with disrupt the membrane structure.
o
Lipid Anchors – Anchored into the membrane using covalently attached hydrophobic groups.
These lipid modifications of proteins include GPI or glycophosphatidylinositol, Farnesylation,
Palmitoylation and Myristoylation. For GPI, if defective, a rare acquired somatic mutation will
cause hemolytic anemia and Thrombosis.
3. Explain the impact of fatty acid composition and cholesterol content on membrane fluidity.
o
Fluidity impacts Protein arrangement, Signaling complex fxns, permeability, with excessive
fluidity leading to membrane destruction.
o
Increased Fatty Acid length = Increased viscosity.
o
Saturation of Fatty Acids = Viscous
o
Desaturation of Fatty Acids= Increased Fluidity
o
Cholesterol disrupts Fatty Acyl side chain interactions.

Below Tm: Increases Fluidity

Above Tm: Decreases Fluidity

Lipid Raft = Cholesterol rich domain of plama membrane
4. Discuss the origins and importance of membrane lipid and membrane protein asymmetry. Describe
the effects of exoplasmic phosphatidylserine.
o
Only the exoplasmic portion of integral membrane proteins are glycosylated. N-linked or Olinked sugars. Happens in the lumen of the ER and Golgi, which determines exoplasmic
glycosylation. Confers specificity, localization and function of protein or lipid.
o
choline–phospholipids = exoplasmic
o
amino–phospholipids = cytoplasmic
o
Cytoplasmic leaflet is enriched in unsaturated fatty acyl chains.
o
Exoplasmic phosphatidyl serine confers macrophage recognition and subsequent destruction.
5. Discuss the interactions among the key protein components of the RBC membrane. Describe their
contribution to its strength and flexibility in health & disease.
o
Hereditary Spherocytosis – 1/5000 North Americans. Determined by the Osmotic Fragility test.
Affected by mutations in either, SPECTRIN, ANKYRIN or other CYTOSKELETAL proteins.
Spherocytotic cells are then subject to destruction in the spleen and microcapillary beds.
Plasma Membrane: Part 2
1. Distinguish the major kinetic difference(s) between simple and facilitated diffusion.
o
Facilitated diffusion utilizes Carrier proteins to accelerate a reaction that is already
thermodynamically favored (utilizing an electrochemical gradient).
2. Compare the ion concentrations in the ECF and ICF and discuss the role of these electrochemical
gradients in membrane transport.
o ΔGion grad + ΔGelect grad = Net ΔGTotal
o Substance
ECF (mM) ICF (mM)
o Na+
140
10
+
o K
4
140
o Cl100
4
2+
o Ca
2.5
.0001
o HCO327
10
o Glucose
5.5
.5
3. Describe the mechanism of action of GLUT4 in insulin-responsive glucose uptake.
o
Insulin binds to RTK receptor, which results in GLUT4 being translocated from the intracellular
vesicular pool to the plasma membrane surface.
4. Describe how the Na+/K+ ATPase primary active transporter functions to drive secondary active
transport of glucose. Define symporter and antiporter.
o
The Na-pump generates and maintains an Electrochemical Gradient. The Sodium that is pumped
into the EC space is used to drive the Secondary transport of Glucose and Na+ to the intracellular
space, through a Na+/Glucose symporter.
o
Symporter is a carrier mediated protein that transports molecules in the same direction. The
coupling of an unfavorable movement of one molecule with the energetically favorable
movement of another.
o
Antiporter is the opposite of a symporter.
5. Explain the mechanism of action of cardiotonic steroid drugs on the Na-pump and the use of digitalis
in heart failure.
o
Inhibits the E2-P dephosphorylation of the Sodium Pump. This leads to increased IC Na+ and
thus increased Ca2+ due to a Na/Ca exchanger. Thus, the increase in IC Ca2+ results in a more
prolonged and efficient contraction of the heart muscle.
6. Explain the structural basis of the ion selectivity of the voltage-gated K+ channel.
o
All ions exist in a hydrated form, each with a distinct size. Thus the channel has size exclusion
properties, wherein only K+ can satisfy for successful passage.
7. Describe the involvement of ABC-type ATP-powered pumps in cystic fibrosis and multi-drug
resistance.
o
The ATP Binding Cassette protein involved in Cystic Fibrosis is defective in pumping Cl- ions
out of the cell. The lack of Chloride ions on the Extracellular face creates a charge differential,
leading to a large influx of Na and subsequently H2O. Thus the mucous that is in the lungs of
Cystic Fibrosis patients is abnormally thick, and difficult to get rid of; making the patient prone to
chronic lung infections. The golden diagnostic for this disease is the sweat Cl- test.
o
Another ABC-type pump is the upregulated MDR1 integral membrane protein. The upregulated
MDR1 actively exports Cancer therapeutics from the cytosol to the extracellular space. Thus the
drug fails to exert its benefits to the Cancer cells. Adriamycin, Doxorubicin, and Vinblastine.
8. Define the basic stages of signal transduction. Explain the involvement of plasma membrane-based
enzymes.
o
Signal  Reception  Transduction  Response/Downstream Amplification.
o
The transduction part of the signal occurs at the Plasma membrane
9. ALWAYS REMEMBER! PLASMA MEMBRANE IS HIGHLY DYNAMIC!!!
Cell Cycle: Dr. Pittman
1. Describe the phases of the cell cycle and the stages of mitosis.
o G0-phase which is a withdrawal phase from the cell cycle; usually highly differentiated cells.
o Interphase – Which is subdivided into G1-gap phase (cell volume restored, RNA metabolism,
regulatory proteins and enzymes necessary for DNA replication), S-synthetic phase
(Replication of nuclear DNA 2n4n, duplicate chromosomes consist of two identical sisterchromatids), and G2-2ndgap phase (Metabolism of RNA more regulatory proteins and
enzymes are synthesized, DNA analyzed for errors w/corrections made).
o Prophase – Chromosomes become condensed and key proteins begin to bind to kinetochores
(center of Sister chromatids), preparing for spindle attachment.
o Prometaphase – Nuclear envelope breakdown, during which the mitotic spindle is formed and
the chromosomes attach to microtubules in the spindle via their kinetochores.
o Metaphase – Maximum condensation of the chromosomes, all of the chromosomes are
attached to microtubules via their kinetochores and aligned at the metaphase plate.
o Anaphase – the sister chromatids separate and are moved toward the poles of the mitotic
spindle.
o Telophase – each set of chromosomes has reached its pole, and the mother cell is physically
divided into two daughter cells by cytokinesis.
2. Explain what checkpoints are, and how they work.
o Checkpoint – A control mechanism that monitors the successful completion of molecular
events in the cell cycle. 1) Environment favorable? 2) DNA damaged? 3) Cell big enough? 4)
Replication complete? 5) Chromosomes aligned properly?
o Cyclins peak at specific stages of the cell cycle, perform their functions by forming
complexes with CDKs, and are rapidly degraded by the ubiquitin-proteasome pathway
(Mitotic cyclin destruction box that associates with ubiq-prot lysine rich region).
o Checkpoints monitor for problems in DNA replication, DNA breaks, and chromosome
segregation. The cell cycle may be delayed until problems are resolved.
o How it works (an example):

Cyclin B is synthesized during the G2 phase.

Cyclin B associates with CDK1 (Cyclin Dependent Protein Kinase 1), which
subsequently autophosphorylates.

The autophosphorylation step is successful if no DNA damage has occurred, etc.

Specifically the Cyclin B complex will regulate Sister Chromatid Cohesion
through the Anaphase Promoting Complex (promotes telophase and polyubiq of
cyclin B, gets rid of the cohesin protein complexes on sister chromatids).
3. Review the experimental systems and approaches that underlie cell cycle regulation studies.
o
Mammalian Cell Fusion Experiments – Heterokaryon fusion, identified diffusible factors such
as S-phase activation factors.
o
Biochemical assays for MPF activity – Provides sources of extracts for biochemical studies
for purification and identification of mitosis promoting factor, which contains Cyclin B.
o
Isolation of yeast cell division cycle mutants – Genetic identification of cell cycle regulators
and their mutated effects. Conditional mutants are temperature sensitive cell division cycle
(cdc) mutants.
4. Summarize the role of cyclins and cyclin-dependent kinases.
o
There are complexes of proteins called cyclins and cyclin dependent kinases (cdks) that
regulate the cell cycle.
o
Cyclins are rapidly synthesized and degraded
o
Cdks are protein kinases that require cyclins for activity
o
Protein kinases phosphorylate a variety of serine, threonin, and tyrosine residue protein
substrates.
5. Associate the cell cycle defects with altered cell growth and division.
o
Nondisjunction – APC will normally prevent cytokinesis if the assembled spindle system is
defective.
o
Phosphorylated RB protein – Retinoblastoma protein is usually phosphorylated, and
subsequently promoting G1S-phase transition via activation of Cyclin D/CDK complex.
However, if either one remains phosphorylated due to a mutation, the cell cycle would
progress out of control. Causing Retinoblastoma.
o
p53 – is required to limit Cellular Growth. Allows time for DNA damage repair, or will
trigger apoptosis via bax mediated pathway.
6. Recognize the clinical significance of uncontrolled cellular proliferation.
o
See above.