BIOCHEM-CHAPTERS 1-5

SAMBISIG TRANSES (FROM HARPERS)

What is the biomedical importance of water?

• Universal solvent/Solvent of life.

• Due to its dipolar structure and capacity to form hydrogen bond.

• Water is reactant or product in many metabolic reactions.

• Excellent nucleophile (an atom or molecule that in chemical reaction seeks a positive center, such as the nucleus of an atom, because the nucleophile contains an electron pair available for bonding).

• Regulation of water balance.

  • Hypothalamic rxns → control thirst.

  • Antidiuretic hormone (ADH) → water retention or excretion by kidneys, and on evaporative loss.

  • Nephrogenic diabetes insipidus → inability to concentrate urine or adjust to subtle changes in extracellular fluid osmolarity, results from the unresponsiveness of renal tubular osmoreceptors to ADH.

• Water tends to dissociate into hydrogen ions (H+) and hydroxide ions (OH-) (H2O ⇌ H+ + OH-).

Define acidity and pH.

• Acidity: Concentration of protons of aqueous solutions - logarithmic scale.

• pH @ homeostasis: pH of extracellular fluid between 7.35 and 7.45.

  • Maintenance by bicarbonate and other buffers.

  • Acid-base balance disturbance.

  • Measuring the pH of arterial blood and the CO2 content of venous blood.

What causes acidosis and alkalosis?

• Acidosis: if blood pH < 7.35 (causes: diabetic ketosis and lactic acidosis).

• Alkalosis: if pH > 7.45 (causes: may follow vomiting of acidic gastric contents).

Why is water an ideal biologic solvent?

• Water molecules form dipoles:

  • Water molecule: irregular, slightly skewed tetrahedron with oxygen at its center.

  • The water molecule has tetrahedral geometry.

  • Two hydrogens and the unshared electrons of the remaining two sp3-hybridized orbitals occupy the corners of the tetrahedron.

  • 105° angle between the two hydrogen atoms differs slightly from the ideal tetrahedral angle, 109.5°.

  • Strongly electronegative oxygen atom in a water molecule attracts electrons away from the hydrogen nuclei → partial + charge.

  • While its two unshared electron pairs constitute a region of local negative charge.

  • Dipole: (molecule with electrical charge distributed asymmetrically about its structure).

• Water → strong dipole → high dielectric constant.

• This result in physical properties of water and accounts for its relatively high viscosity, surface tension, and boiling point.

• Also enable water to dissolve biomolecules containing functional groups (aldehydes, ketones, and amides - hydrogen acceptors).

• Alcohols, carboxylic acids, and amines - both as hydrogen acceptors and as donors.

How do water molecules form hydrogen bonds?

Hydrogen Bond: dipole-dipole interaction between a hydrogen atom that is bonded to a highly electronegative atom and another highly electronegative atom.

How do Covalent and Noncovalent Bonds Stabilize Biologic Molecules?

Covalent bond is the strongest force that holds molecules.

What are Acids and Bases, and how is pH defined?

• The term pH was introduced in 1909 by Sørensen, who defined pH as the negative log of the hydrogen ion concentration.

• pH = -log ([H+])

• Low pH: High H+.

• High pH: High OH- (low H+).

• Acids: proton donors.

• Bases: proton acceptors.

• The stronger the acid, the lower is its pKa value.

• Strong acids/bases completely dissociate in low pH/high pH respectively. Weak acids/bases do not.

Describe functional groups that are weak acids and have great physiologic significance.

• Many biochemicals possess functional groups that are weak acids or bases.

• Protonated species (R—NH3+) are referred to as acids, unprotonated as conjugate base (R—NH2).

• Or, base (R—NH2) and conjugate acid (R—NH3+).

• Solutions and their conjugates experience buffering (resistance to change in pH) after addition of a strong acid or base.

• Since the numeric values of Ka for weak acids are negative exponential numbers, we express Ka as pKa, where pKa = -logKa.

What is the Henderson-Hasselbalch Equation and what is its purpose?

• Purpose: To determine the pH of weak acids and buffers.

• Equation: pH = pKa + log([A-]/[HA])

How is pH maintenance achieved in biochemical processes?

Most biochemical processes are buffered. pH maintenance involves buffering by proton release from phosphate, bicarbonate, and proteins.

How does acid strength depend on molecular structure and medium?

• Many acids of biologic interest possess more than one dissociating group, hindering proton release from nearby groups and raising pKa.

• The medium may either raise or lower the pKa, depending on whether the undissociated acid or its conjugate base is the charged species (presence of ethanol, water, etc.).

What is the biomedical importance of biochemistry?

Biochemistry illuminated various aspects of health; diseases opened up new areas of biochemistry.

What are the mutual advantages provided by biochemistry and medicine?

• 2 major concerns in health sciences:

  • Understanding maintenance of health.

  • Effective treatment of diseases.

• 2-way street of biochemistry & medicine.

What early medical discoveries illuminated by biochemistry are mentioned?

• Sickle cell Hgb: Caused by a single difference in amino acid from normal Hgb. Further analysis of other variants and other Hgbs lead to deeper understanding of Hgb structure & function and of other proteins.

• Archibald Garrod: Studied patients with alkaptonuria, albinism, cystinuria and pentosuria, designated these conditions as inborn errors of metabolism.

How has biochemistry understanding aided by medical diseases?

Investigation of familial hypercholesterolemia that results in early-onset atherosclerosis. This led to the understanding of cell receptors and mechanisms of uptake of molecules across cell membranes.

What biochemical processes underlie human health?

• Biochemical Research Impacts Nutrition & Preventive Medicine.

• Health: "complete physical, mental, and social well-being and not merely the absence of disease and infirmity." (WHO)

• Maintenance: dietary intake of vitamins, certain amino acids and fatty acids, various minerals, and water.

• Understanding of biochem=understanding of nutrition.

• Preventive medicine: attempts to maintain health and forestall disease; includes nutritional approaches to prevention of diseases (e.g. Atherosclerosis and tennis).

What is the impact of the Human Genome Project on biochemistry, biology, & medicine?

• 50 yrs after DNA nature description by Watson & Crick, human genome was sequenced completely in 2003 by the International Human Genome Sequencing Consortium and by Celera Genomics.

• Genetic manipulation - can potentially provide clues to cure human diseases such as Alzheimer's & cancer.

• Studies of oncogenes and tumor suppressor genes lead to focusing on normal cell growth molecular mechanisms.

What is the biomedical importance of L-α-amino acids?

• Monomer units of protein polypeptide chains.

• Participate in diverse cellular function:

  • Nerve transmission.

  • Biosynthesis of:

    • Purines

    • Pyrimidines

    • Urea

How are L-α-amino acids involved in the Neuroendocrine system?

Employs:

• Peptides as hormones.

• Hormone releasing factors.

• Neuromodulators.

• Neurotransmitters.

What are nutritionally essential amino acids?

• 10 of the L-α-amino acids present in proteins.

• Essential for:

  • Infant growth

  • Adult health maintenance

• Cannot be synthesized by humans and other higher animals.

• Important to be included in the human diet.

Explain amino acid conservation in the body.

• 50 g free amino acids are filtered, from the arterial renal blood to kidney.

• Only traces of free amino acids are found in the urine.

• Amino acids are reabsorbed in the proximal tubule.

• Conserved for protein synthesis and other functions.

How do microorganisms utilize amino acids?

• May secrete free D-amino acids or peptides.

• May contain both D- and L-α-amino acids.

Give examples of bacterial peptides with therapeutic value.

• Antibiotic: bacitracin, gramicidin A.

• Antitumor agent: bleomycin.

Describe toxic microbial peptides.

Cyanobacterial peptides:

• Microcystin: Large dose → lethal; small dose → promote hepatic tumor formation.

• Nodularin: Large dose → lethal; small dose → promote hepatic tumor formation.

What are the effects of certain amino acids found in seeds?

• Seeds of legume in genus Lathyrus: lathyrism → irreversible disease, loss of limb control.

• Other plant seed amino acids → neurodegenerative disease in Guam natives.

What are the general properties of amino acids regarding the genetic code?

Back:

• The Genetic Code specifies 20 L-α-amino acids.

• There are more than 300 amino acids in nature.

• Codons: nucleotide triplets that forms amino acids.

What are the four groups attached to the α-carbon of an amino acid?

• An acidic carboxyl group (-COOH).

• A basic amino group (-NH2).

• A hydrogen atom (-H).

• A distinctive side chain (R-group).

What is the significance of the α-carbon in amino acids?

• In all L-α-amino acids except glycine, the α-carbon is asymmetric (chiral).

• This means amino acids are optically active.

• All protein L-α-amino acids have positive, negative or zero net charges.

• Amphoteric.

• Zwitterion.

List some common hydrophilic amino acids.

Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Lysine, Serine, Threonine.

List some common hydrophobic amino acids.

Alanine, Isoleucine, Leucine, Methionine, Phenylalanine, Proline, Tryptophan, Tyrosine, Valine.

What are Ornithine and Citrulline?

Key intermediates → urea cycle.

What are L-α-amino acids vital metabolic roles?

• Tyrosine is a precursor of thyroid hormone, norepinephrine, and epinephrine.

• Tryptophan is converted to serotonin and niacin.

• Ornithine and Citrulline are intermediates in urea biosynthesis.

• Glycine, aspartate, and glutamine are intermediates in the metabolism of the protein amino acids.

Give examples of certain plant L-α-Amino Acids that can adversely impact human health.

• Lathyrus: neuro-lathyrism - a neurological disorder characterized by progressive and irreversible spastic paralysis of the legs.

• Homocarginine and B-N-oxalyl-L-α,B-diaminopropionic acid (B-ODAP) have been implicated in human neurologic disorders.

• 'Sweet pea' - contain osteolathyrogen γ-glutamyl-B-aminopropionitrile (BAPN), a glutamine derivative of B-aminopropionitrile.

• Another seed from Lathyrus species - contains α,γ-diaminobutryic acid - analog of ornithine that inhibits the hepatic urea cycle enzyme ornithine transcarbamylase, leading to ammonia toxicity.

• Cycad seeds → contains 'B-methylaminoalanine' - neurotoxic amino acid has been implicated as a risk factor for neurodegenerative diseases including amyotrophic lateral sclerosis - Parkinson dementia complex in native in Guam who consume fruit bats that feed on cycad fruit and or flour made from cycad seeds.

Describe D-Amino Acids.

• Occurs naturally in the biosphere.

• Free D-serine and D-aspartate → human brain tissue.

• D-alanine and D-glutamate → cell walls of gram positive bacteria.

• D-amino acids → peptides and antibiotics from bacteria, fungi, reptiles, and amphibians.

• Bacillus subtilis - secretes D-methionine, D-tyrosine, D-leucine, and D-tryptophan → formation of biofilm assembly.

• Vibrio cholerae - D-leucine and D-methionine → peptide component of its peptidoglycan layer.

Describe the stereochemistry of protein amino acids.

• A-α-carbon of every amino acid is chiral (except glycine).

• Some protein amino acids are dextrorotatory and some levorotatory.

• All share absolute configuration of L-glyceraldehyde and are defined as 'L-α-amino acid'.

What are posttranslational modifications?

• Some prokaryotes incorporates pyrrolysine into proteins.

• Plants can incorporate azetidine-2-carboxylic acid (analog of proline).

• Collagen (protein bound residues of proline and lysine) → converted to 4-hydroxyproline and 5-hydroxylysine.

• Carboxylation of glutamyl residues of proteins of the blood coagulation cascade to γ-carboxyglutamyl residues → complete a site for chelating calcium ion essential for blood coagulation.

• Amino acid chains of histone are subject for modifications - including acetylation and methylation of lysine and methylation and deimination of arginine.

What is the significance of Extraterrestrial Amino Acids in Meteorites?

• Extraterrestrial Amino acid from Murchison meteorite from Southern Australia in 1969.

• These meteorites contain racemic mixtures of D- and L-isomers of multiple protein amino acids, non-protein α-amino acids (N-methylglycine (sarcosine) and B-alanine).

• 'Panspermia' - delivering extraterrestrial generated organic molecules or even intact microorganisms on earth.

What is the role of L-α-Amino Acids?

Serving as the "building blocks" of proteins.

Describe Selenocysteine, the 21st Protein L-α-Amino Acid.

• Humans contain two dozen of selenocysteine that includes certain peroxidases and reductases.

• Selenoprotein P: circulates in the plasma.

  • Responsible for converting prohormone thyroxine (T4) to the thyroid hormone 3,3', 5-triiodothyronine (T3).

• tRNAsec incorporated selenocysteine → utilizes UGA anticodon.

What are the properties of amino acids regarding light absorption?

• Amino acids do not absorb visible light and thus colorless.

• Tyrosine, phenylalanine, tryptophan - absorbs high wavelength (250-290nm) Ultraviolet light.

How do α-R groups determine the properties of amino acids?

• Carboxylic acid groups: formation of ester, amides, acid anhydrides.

• Amino groups: acylation, amidation, esterification.

• -OH and -SH groups: oxidation, esterification.

• Glycine: often occurs where peptides bend sharply.

• Hydrophobic R-groups (alanine, valine, leucine, isoleucine) & aromatic R-groups (phenylalanine, tyrosine, tryptophan): primarily in the interior of cytosolic proteins.

• Charged R-groups (basic & acidic amino acids): stabilize specific protein conformation via ionic interactions or salt bridges.

• Primary structure: number and order of amino acid residues in a polypeptide constitute.

Describe aminoacyl residues in peptides.

• Aminoacyl residues → amino acids present in peptides, replacing 'ate' or 'ine' suffixes of free amino acids with 'yl'.

• 'Tri' or 'octa' → peptides with three to eight residues.

• Peptides are drawn using a zigzag manner to represent the main chain or the backbone.

How are amino acids linked in mammals?

• Mammals - 20 codon-specified amino acids linked by standard peptide bonds (may contain non-protein amino acids or amino acids linked by an atypical peptide bond).

What are the properties of functional groups of amino acids regarding charge?

• Amino Acids May Have Positive, Negative, or Zero Net Charge.

• R-COOH ⇌ weak acid, stronger than R-NH3.

• R-NH3 ⇌ weak acid.

• In aqueous solution, the charged and uncharged forms of the ionizable weak acid groups -COOH and -NH3+ exist in dynamic protonic equilibrium:

  • R-COOH ⇌ R-COO- + H+

  • R-NH3+ ⇌ R-NH2 + H+

• At pH 7.4, the α-carboxyl groups of free amino acids exist almost entirely as R-COO- and α-amino groups predominantly as R-NH3+.

• "Zwitterions" - ion possessing equal numbers of positively and negatively charged groups.

• Structure A (amino acids in blood and tissues) - zwitterion.

• Structure B - cannot exist in aqueous soln pH low enough to protonate the carboxyl group, amino group would also be protonated.

How are pKa values expressed for weak acids and bases?

• pKa ⇌ strength of weak acids.

• Subscript 'a' replacing with a number.

• Strength of weak bases → expressed as the pKa of their protonated or conjugate acid form.

• The net charge of the amino acid depends on the pKa values of the functional groups and the pH of the surrounding medium.

What is the isoelectric pH (pI) of an amino acid?

At its isoelectric pH (pI), an amino acid bears no net charge.

• Isoelectric species: zwitterions.

• Isoelectric point (pI): pKa; pI pH midway between pKa values for ionizations on either side of the isoelectric species.

• Knowledge of pI for electrophoretic separations.

How do pKa values vary with the environment?

• The environment of a dissociable group affects its pKa.

• NONPOLAR ENVIRONMENT: ↑pKa of carboxyl group (weaker acid); ↓pKa of amino group (stronger acid).

• The presence of an adjacent oppositely charged group can stabilize or can destabilize a developing charge.

What does the solubility of amino acids reflect?

Their ionic character.

Describe the peptide bond.

• Bond connects a carbonyl carbon to the α-nitrogen - cannot rotate - breaking the partial double bond.

• O,C,N, H → coplanar.

• Peptide bond is uncharged at any pH of physiologic interest.

• For amino acids, net charge on a peptide depends on the pH of its environment and on the pKa values of its dissociating groups.

• Peptides bond has partial double-bond character.

What is the biomedical importance of proteins?

• Proteins are complex macromolecules that perform critical roles.

• Cytoskeleton: maintains the cell shape.

• Actin and myosin: muscles' contractile machinery.

• Hemoglobin: oxygen transporter.

• Enzymes: biological catalyst.

• Receptors: enable cell to sense and respond to hormones.

• Typical Proteins: born at posttranslational matures through posttranslational processes (selective proteolysis).

• Molecular Medicine: identify biomarkers (e.g. proteins, changes in protein structures, deficiency that corresponds to specific diseases).

Why must proteins & peptides be purified prior to analysis?

Highly purified proteins are essential for detailed examination.

What are the methods for selective precipitation of proteins?

Differences in solubility as a function of pH, polarity (precipitation with ethanol and acetone) or salt concentration (salting out with NH4SO3).

Size-exclusion/gel filtration chromatography

based on size. Separates protein base on stroke radii.

Ion-exchange chromatography

net charge. Proteins interact with stationary phase by charge-charge interactions. Opposite charge attracts. No adherent proteins will be washed away.

Hydrophobic interaction chromatography

ability to attach with stationary phase coated with hydrophobic groups.

Affinity chromatography

ability to bind with specific ligand.

Column chromatography

stationary phase matrix consist of beads in the cylindrical container called "column". A liquid-permeable glass confines the bead in the column as mobile-phase liquid flow through. Mobile-phase liquid appears from the column, it collects "fraction"- series of small potion.

HPLC

• consisted of long, intertwined oligosaccharide polymers shaped into spherical beads.

How is protein purity assessed by Polyacrylamide Gel Electrophoresis (PAGE)?

• Electrophoresis: separates charged biomolecules by the rate of migration in an applied electrical field.

• SDS-PAGE: most widely used. PAGE incorporated with anionic detergent (sodium dodecyl sulfate).

• Protein migrates as SDS-polypeptide complexes through polyacrylamide matrix. Trapped polypeptides on gel will be stained by Coomassie Blue.

Who devised the first practical method for peptide sequencing?

Edman.

Describe Sanger's method for sequencing a polypeptide.

• Sanger was the first to determine the sequencing of a polypeptide.

• Insulin: First peptide hormone.

• Trypsin, Chymotrypsin and pepsin: cleaved smaller peptide chains after breaking the disulfide bond of insulin.

• 1-fluoro-2,4-dinitrobenzene (Sanger reagent): treated the small peptide chains.

• Determines 20 amino acids.

• Developed dideoxy method for DNA sequencing.

How has molecular biology revolutionized the determination of primary structure?

• Recombinant techniques: manufactures a virtually infinite supply of DNA from even minute quantities of template present.

• Better than Sanger's and Edman's method.

How does genomics enable proteins to be identified from small amounts of sequence data?

(No specific details provided in the image, just that it does enable this.)

How can mass spectrometry detect covalent modifications?

• Replaced Edman's techniques for protein identification.

• Separates peptide based on their mass differences.

What are the common configurations of mass spectrometers?

• Single Quadrupole MS: molecules with a mass of 4 kDa or less.

• Sample place under the vacuum and vaporize in the presence of proton donor to impart positive charge.

• Electrical field propels cations to flash tube and encounter magnetic field.

• Ions of identical net charge-force required to bend their path is proportionate to their mass.

• Time-of-flight MS: linear flight tube. After vaporization took place, electric field is applied to accelerate the ions to a detector at the end of the flash tube.

  • For identical net charge: velocity and time is proportional to mass.

  • Determines large masses of proteins.

How can peptides be volatilized for analysis by electrospray ionization or matrix-assisted laser desorption?

• Tandem MS: can analyze protein without prior purification.

  • MS-MS

  • Screens blood of a newborn for possible diseases (PKA, ethylmalonic encephalopathy, glutaric academia type 1).

  • New MS-MS: Q-TOF-MS.

• 3 common methods:

  • Electrospray ionization: sample is dissolved in a solvent.

  • Probe: ionizes the sample and disperses the solvent.

  • Matrix-Assisted Laser Desorption and Ionization (MALDI): sample + liquid matrix with light-absorbing dye.

  • Fast Atom Bombardment: large.

• MACROMOLECULES: disperse in glycerol.

What is the goal of Proteomics?

To identify the Entire Complement of Proteins Elaborated by a Cell under Diverse Conditions.

• As genes are switched on and off, proteins are synthesized in particular cell types at specific times of growth or differentiation and in response to external stimuli.

• Proteome: a term that refers to the identity, abundance, and state of modification of the entire suite of proteins expressed by an individual cell at a particular time.

What are the challenges in the simultaneous determination of hundreds of proteins?

Technically challenging.

Describe First Generation Proteomics.

Uses SDS-PAGE or two-dimensional electrophoresis to resolve the proteins in a biologic sample from the other, and then determination if amino acid from the terminus using Edman's method.

Describe Second Generation Proteomics.

• Nanoscale chromatographic techniques with MS.

• Proteins + protease to hydrolyze to smaller peptides then subjected to ion exchange, reversed phase or size exclusion chromatography.

• Multidimensional Protein Identification Technology (MudPIT).

  • Resolves peptides produced to simple fraction that can be analyzed by MS.

How does bioinformatics assist in the identification of protein functions?

Development of computer algorithms to infer the functional properties of macromolecules through comparison of sequences of new proteins with the properties of unknown.

What is the biomedical importance of protein conformation?

• Conformation follows function: Proteins must fold into a specific three-dimensional conformation.

• Genetic or nutritional deficiencies that impede protein maturation are deleterious to health.

Differentiate between Conformation and Configuration.

• Configuration: the geometric relationship between a given set of atoms.

• Conformation: the spatial relationship of every atom in a molecule.

What are the four orders of protein structure?

• Primary Structure: amino acid in a polypeptide chain (linear, no folding).

• Secondary Structure: Folding of short amino acid (3-30 residues) into geometrically ordered structure (with folding).

• Tertiary Structure: Assembly of secondary structure into larger three-dimensional functional units—a mature polypeptide chain with domains; how secondary structural features (helices, sheets, bends, turns, and loops) assemble to form domains.

• Quaternary Structure: Arrangement of 2 or more polypeptide chains which may be related or unrelated. (Lippincott) It defines the polypeptide composition of a protein and spatial relationships for oligomeric proteins.

Describe the Alpha Helix secondary structure.

• Backbone is twisted by the ff angles:

  • Phi angle (Φ) = –57°, Psi angle (Ψ) = –47°.

  • R groups face outward.

• Comprised only solely of L-Amino acid, only stable form they can form are right-handed.

• Proline: lacks hydrogen atom, cannot form hydrogen bond (only during first turn), can disrupt helix formation and produce a bend.

• Glycine: very short R group, can disrupt packing and introduce bend within the helix.

• Amphipathic helices create channels and pores in cell membranes.

Describe the Beta Sheet secondary structure.

• Backbone is highly extended; zigzag/pleated pattern from the edge.

• Same stability with alpha helix.

• Bonds are formed between adjacent segments of β sheet.

• Adjacent segments in same direction – parallel.

• Adjacent segments in opposite direction – antiparallel.

• Clusters of β sheets (β barrels) form core of many globular proteins.

Describe Loops & Bends in protein structure.

• Short segments of amino acids that join 2 units of secondary structure.

• Loops: regions containing residues beyond minimum number to connect adjacent regions of secondary structure; some residues in loops can bridge domains for enzyme binding sites.

What are Helix-Loop-Helix motifs?

Provide oligonucleotide-binding portion to many DNA-binding proteins.

What are Supersecondary structures?

Intermediate between secondary and tertiary structures.

What are protein domains?

• A section of protein structure sufficient to perform a particular chemical/physical task (e.g. binding of substrate or other ligands).

• Not all domains bind substrates, hydrophobic domains anchor proteins and allows them to span membranes.

• Regulatory domains trigger changes in protein function in response to effectors or other modifications.

Differentiate between types of proteins based on quaternary structure.

• Monomeric proteins: single polypeptide chain.

• Dimeric proteins: 2 polypeptide chain.

• Homodimers: 2 copies of same polypeptide chain.

• Heterodimers: 2 copies of different polypeptide chain.

What multiple factors stabilize tertiary and quaternary structures?

• Noncovalent bonds stabilize higher protein orders primarily and almost exclusively. Hydrophobic interactions protein structure from water.

• Hydrogen bonds and salt bridges between carboxylates of some amino acids and protonated amino acids.

• Some proteins contain covalent disulfide (S-S) bonds that link sulfhydryl of cysteinyl residues which enhances stability of folded conformation of peptide.

How do biophysical techniques reveal three-dimensional structure?

• X-RAY CRYSTALLOGRAPHY:

  • Protein is precipitated to form crystals.

  • Crystals irradiated with X-ray beams and pattern formed are diffracted → patterns formed present as series of spots on film → to calculate position, phases of diffracted protein will first be determined.

• Isomorphous displacement: atom with strong X-ray signature (e.g. mercury or uranium) is introduced into a crystal at known positions in the primary structure (to determine protein position: the unknown structure should be analyzed with known structures to compare identity and structure).

• NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY: measures absorbance of radio frequency by certain atom nuclei.

• CRYO-ELECTRON MICROSCOPY: employs ultracold media (e.g. liquid nitrogen, ethane, or helium) to stabilize biomolecules.

• MOLECULAR MODELING:

  • Molecular Dynamics: simulation of conformational dynamics of a protein under certain conditions.

  • Molecular Docking: simulation of interactions when protein encounters substrates, inhibitors, or other ligands.

• Homology Modeling: known 3D model of protein is used as template to make a probable structure of a related protein.

How may perturbation of protein conformation have pathologic sequences?

• PRION DISEASES (Transmissible Spongiform Encephalopathies):

  • Fatal neurodegenerative diseases (spongiform changes, astrocytic gliomas, neuronal loss).

  • Creutzfeldt-Jacob Disease (vCJD): early-onset psychiatric and behavioral disorders.

• ALZHEIMER'S DISEASE:

  • High levels of β-amyloid which undergoes a conformational transformation.

• BETA-THALASSEMIA: Genetic defects that impair synthesis one polypeptide subunit of hemoglobin.

What illustrates the role of posttranslational processing in protein maturation?

• COLLAGEN.

• Maturation of proteins involves cleavage of formation of covalent bonds.

What are proproteins?

Proproteins are larger precursors that undergo posttranslational modification.

Describe Collagen as a fibrous protein.

• Collagen, keratin, myosin - 25% of muscle protein mass in body.

• For strength and flexibility of skin (along with keratin).

• Present in ligaments and tendons.

• Has high degree of tensile strength.

How does Collagen form a triple helix?

• Elongated rod with axial ratio of 200.

• Every 3 polypeptide strands twist to the left, wrap around another right making the helix highly resistant to unwinding.

How is collagen synthesized and activated?

• Collagen is synthesized as a larger precursor, initially synthesized as procollagen.

• Activation is by enzymes that require Vitamin C (and copper).

What nutritional & genetic disorders can impair collagen maturation?

• Scurvy: Vitamin C deficiency; impaired collagen activation and collagen fiber stability - bleeding gums, swelling joints, poor wound healing.

• Menkes Syndrome: Copper deficiency; kinky hair growth.

• Ehlers-Danlos Syndrome: connective tissue disorder due to genetic disorder of collagen synthesis.

What are the general properties of an amino acid structure?

• Four different groups connected to a tetrahedral alpha-carbon atom.

• Alpha-carbon is chiral (except in glycine).

• Composed of an amine group (NH2​), a carboxyl group (COOH), a hydrogen atom, and a side chain (R group).

How many amino acids are "of significance" in nature and how many occur exclusively in proteins?

• Over 300 amino acids in nature are of significance.

• Only 20 L-amino acids occur exclusively in proteins.

What type of amino acids occur in proteins (L- or D-)?

Only L-amino acids

What kind of charge can amino acids have?

Positive, negative, or zero net charge.

Are amino acids strong or weak acids/bases?

Strong acids/weak acids.

Describe the solubility characteristics of amino acids based on their R groups.

Can be hydrophilic or hydrophobic.

What is a zwitterion?

An amino acid can exist as a zwitterion, a molecule or ion having separate positively and negatively charged groups.

List the amino acids with Aliphatic Side Chains.

• Glycine [Gly] [G]

• Alanine [Ala] [A]

• Valine [Val] [V]

• Leucine [Leu] [L]

• Isoleucine [Ile] [I]

List the amino acids with Side Chains Containing Hydroxyl (OH) Groups.

• Serine [Ser] [S]

• Threonine [Thr] [T]

• Tyrosine [Tyr] [Y]

List the amino acids with Side Chains Containing Sulfur Atoms.

• Cysteine [Cys] [C]

• Methionine [Met] [M]