Acid / Base chemistry is crucial for living organisms (ph control and acid/base catalysis) water as an acid, donates a proton.

Transcription

1 hapter 2: Protein tructure and Function Acid / ase chemistry is crucial for living organisms (p control and acid/base catalysis) Water as the reference acid with a generic base, :. Water as the reference base with a generic acid A. A water as an acid, donates a proton K b and pk b water as a base, accepts a proton K a and pk a urved arrows emphasize electron movement. ydroxide is the conjugate base of water ydronium ion is the conjugate acid of water A K eq = [+ ][A: ] [ 2 ] -log (K a ) = -log [ + ] - log [A: ] pk a = p - log [A: ] G =.4 pk a pk a (kcal/mole) K a = K eq [ 2 ] = [+ ][A: ] G = - 2.3T logk a = 2.3T (-log K a ) = 2.3T (pk a ) G = (constant)(pk a ) p(x) = -log (x) when [A: ] = pk a = p - G K eq = T G = 5.8 pk a 6 pk a (kj/mole) [ 2 ] 55.5 M T = 300 K assumptions pk =-(log K) (by definition) = 2.0 cal/(mol-k) = 8.4 joule/(mol-k) Also true. G = - T G = free energy bond energies probabilities (randomness) Weak acids ( 2,,, 3 +, 3 P 4, 2 P4 -, P 4-2, 2 3, 3 -, etc.) weaker acid & base (more stable) Y Y stronger acid & base PE weaker acid Y + Y - T stronger base endergonic Y G = P = progress of reaction G =.4 pk a pk a (kcal/mole) G = 5.8 pk a 6 pk a (kj/mole) The equilibrium shifts towards the weaker conjugate acid and base (away from the stronger acid and base). Weaker is more stable (think "less reactive"). trong acids (l, r, I, 2 4, 3, etc.) stronger acid & base A A weaker acid & base (more stable) PE potential energy + - A stronger T acid A weaker base G = exergonic A P = progress of reaction 2

2 Amino Acids with onpolar "" groups (have two pk a 's), All aa chiral centers are except cysteine (because of the sulfur) pk a =2.35 pk a = glycine = name Gly = 3 letter code G = letter code pk a =9.76 pk a = isoleucine Ile I pk a = pk a = methionine Met M pk a =2.35 pk a =2.6 3 alanine Ala A 2 pk a = pk a =9.8 3 phenylalanine Phe F 3 body p 7.4 pk a =2.29 pk a =2.43 pk a = valine Val V pk a = trytophan Trp W ome amino acids have an additional pk a. K a pk a 3 K a2 pk a2 pk a =9.74 pk a = leucine 3 Leu L pk a =.95 ur bodies need 20 amino acids to make our proteins (maybe 22 with some selenium variations). proline Pro P 2 pk a = Amino Acids with Polar "" groups body p 7.4 pk a =9.2 pk a =9.0 pk a =2.9 3 pk a = serine threonine er pk a 3 Thr pk a 3 T pk a =0.3 pk a = pk a =9. 3 tyrosine Tyr Y 3 2 pk a =2.9 cysteine ys 2 3 dimer 2 cystine = 2 x cysteine with disulfide linkage pk a = pk a =8.33 pk a 5 pk a =2. 2 pk a = asparagine Asn 2 pk a 5 pk a = pk a =9.3 3 glutamine Gln Q ther relevant biological pk a values phosphoric acid -2 3 P P 4 P 4 P 4 pk a =2. pka =7.2 pka =2.4 carbonic acid pk a =6.4 pka =

3 Amino Acids with harged "" groups (have three pk a 's) body p 7.4 pk pk a =.99 a =9.90 pk a =9.47 pk a =9.8 pk a = pk a = pk a = pk 2 a = asparatic acid glutamic acid 3 2 pk a =3.90 lysine Asp Glu Lys D E K pk a = pk a = pk a = arginine Arg pk a =.80 pk a = histidine is pk a =9.33 pk a =2.9 cysteine ys Essential AAs istidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine pk a = pk a =8.33 onessential AAs Alanine Arginine Asparagine Aspartic acid ysteine Glutamic acid Glutamine Glycine Proline erine Tyrosine elenocysteine rnithine All amino acids are "" absolute configuration at the position, except cysteine (because the sulfur atom changes the order of priorities). Isoleucine (3) and theonine (3) have a second chiral center. These are the starting points for our body's proteins. Their pk a 's can change in an actual protein invironment due to nearby hydrophobic, hydrophilic and/or ionic groups. 5 enderson-asselbach Equation [A ] p = pk a + log extracellular blood p 7.4 intracellular 6.8 stomach small intestines 8.5 What do the amino acids look like? pk a = p when = [A: ] p = pk a 2 pk a , = 2 + log [A ] 7.4 = log Typical aa ammonium Typical aa carboxylic [A + ] acid ionization constant acid ionization constant log log = ( ) = -2.0 [A ] = (7.4-2) = 5.4 [A + ] = 0 [A ] = / 00 = 0 = 2.5x0 5 = 250,000 / [A + ] pk a 0.8 pk a asparatic acid lysine 2,500 2,500 pk =0 and a 0.79 (third pk glutamic acid a ) 7.4 = log 7.4 = 4 + log [A ] [A + ] (second pk a ) log = ( ) = -3.4 [A + ] log [A ] = (7.4-4) = 3.4 = = / 2,500 [A [A ] ] = 0 = 2.5x0 3 = 2,500 / pk a 2. pk a =7.2 pk a =2.4 3 P 4 2 P 4 2 P 4-2 P P 4 P 4 200,000 ratio = ,

4 enderson-asselbach Equation [A ] p = pk a + log extracellular blood p 7.4 intracellular 6.8 stomach small intestines 8.5 What do the amino acids look like? pk a = p when = [A: ] p = pk a Important acid/base catalyst 2 26,000 histidine and binds with metals 2 is pk a = log arginine [A + ] pk pk a =6.04 a =2.48 log = (second pk a ) (third pk a ) [A + ] ( ) = = 0-5. = / 26,000 [A 7.4 = 6 + log + ] [A + ] pk a 0. log [A ] = (7.4-6) =.4 tyrosine [A ] = 0.4 = 2.5x0 pk a =0. = 25 / (third pk a ) = 0. + log [A + ] Any amino acid pk a value can be shifted, left or right by its enzyme environment. More log = (7.4-0.) = -2.7 [A + ] hydrophobic regions will favor the neutral forms ( = = / 500 2, 2 ). A nearby opposite [A + ] charge will favor the ionic form (nearby pk a 8 pk positive favors negative and vice versa). An a 3 cysteine open environment that allows access to water pk a =8.33 serine is similar to the reference aqueous values (second pk a ) threonine 8 340,000 (obtained in water). It is therefore hard to pk a = log [A ] (third pk a ) determine the form of a functional group 7.4 = 3 + log [A ] (ionic or neutral) in a particular region of a log = protein without knowing something about its ( ) = -0.9 [A + = ] log (7.4-3) = -5.6 [A + ] structure. = = / 8 [A + = = / 340,000 ] [A + ] pk a= pk a= enderson-asselbach Equation extracellular blood p 7.4 intracellular 6.8 [A ] p = pk a + log stomach small intestines 8.5 What do the amino acids look like? pk a = p when = [A: ] p = pk asparatic acid a 4 Typical aa ammonium pk a 9.4 acid ionization constant and glutamic acid (second pk a ) 00 pk 2,500 a 5 pk a 9.4 ratio = 2500/ ratio = /00 If K a gets smaller: pk a gets??? larger 2 4 pk a 5 ratio = 250/ pk a 6 ratio = 25/ pk a 7.4 ratio = / pk a 8.4 ratio = /0 pk a 0.4 ratio = /,000 pk a.4 ratio = /0,000 2 reference = pk a 4.7 ompare to reference, pk a gets a. igher b. Lower c. o change 2 pk a 4.9 pk a pk a pk a 2.3 pk a pk a 7.6 pk a pk a

5 What happens to the pk a when... There is nearby negative charge? pk a? a. pk a is higher b. pk a is similar c. pk a is lower There is a hydrophobic pocket? pk a? a. pk a is higher b. pk a is similar c. pk a is lower There is nearby positive charge? pk a? a. pk a is higher b. pk a is similar c. pk a is lower 9 What happens to the pk a when... There is nearby negative charge? pk a? 3 2 a. pk a is higher b. pk a is similar c. pk a is lower There is a hydrophobic pocket? 3 pk a? 2 a. pk a is higher b. pk a is similar c. pk a is lower There is nearby positive charge? pk a? 3 2 a. pk a is higher b. pk a is similar c. pk a is lower 0 5

6 ecause of resonance of the nitrogen lone pair with the = the amide bonds are planar. This is called the peptide bond. = single bond conformation (trans or cis) resonance resonance trans conformation is favored 000/ over cis. cis steric crowding,000...to... flat flat flat flat flat The flat shape of the amide bond limits possible conformations of proteins. 80 o amachandran Plot α - α distance when trans is 3.8 A and when cis 2.9 A (more crowded). 0 o = x o = y o = 80 o = y o -80 o -80 o = alpha helix = beta pleated sheets 0 o 80 o =x o riginal outlines in f (phi) and y (psi) space proposed by G.. amachandran in 963. olid lines enclose region allowed by hard-sphere bumps at standard radii; dashed lines show region allowed with reduced radii; dotted lines add region allowed when the tau angle (-alpha-) is relaxed slightly. 2 6

7 Proline is unusual in that both trans and cis conformations are possible. It is referred to as a disrupter of normal protein patterns ( helices and pleated sheets). trans favored over cis 3/ in proline (also depends on tensions in the protein chain) ormally, this is an crowded trans 2 3 ormally, 2 is an cis 3 also crowded terminus (#) 3 Proteins are polymers. Less than 40 amino acids are considered peptides. Their specific spatial conformations are controlled by ionic interactions, hydrogen bonding, dispersion forces and disulfide linkages. The most common ways to determine their 3D structure are X-ray crystallography and M spectroscopy. The amino end is referred to as the -terminus and the carboxyl end is referred to as the -terminus. ounting residues always starts at the -terminus (- + 3 ) and finishes at the -terminus ( ). The primary structure of a protein is determined by the gene sequence in DA, as transcribed to the A, as tranlated into the protein (with the possibility of post translational modifications, which cannot be determined from the DA sequence). Primary protein structure = the linear order of amino acids. 3 2 alanine serine phenylalanine tyrosine cysteine -terminus (#n) Possible variety = (20) n n= (20 choices) n=2 (400 choices) n=3 (8,000 choices) n=4 (60,000 choices) n=00 (20 00 choices) 4 7

8 econdary structure refers to highly regular local sub structures on the actual polypeptide backbone chain. Two main types of secondary structure, are alpha helices and beta pleated sheets. In 95 Linus Pauling suggested both alpha helices and beta sheets as a way of maximizing all the hydrogen bond donors and acceptors in the peptide backbone. 5 β pleated sheets can be parallel or anti parallel. 6 8

9 3 3 #n # # #n # #n # #n Tertiary tructure Tertiary structure refers to the three dimensional structure of monomeric and multimeric protein molecules. The alphahelices and beta pleated sheets are folded into a compact globular structures. The folding is partly driven by the non specific hydrophobic interactions, the burial of hydrophobic residues from water, but the structure is stable only when the parts of a protein domain are locked into place by specific tertiary interactions, such as salt bridges, hydrogen bonds, and the tight packing of side chains and disulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol (intracellular fluid) is generally a reducing environment. AD can reduce the disulfide bond to 2 thiols. -pleated sheets -heliz random strands 8 9

10 Quaternary structure refers to the three dimensional structure of a multi subunit protein and how the subunits fit together. The quaternary structure is stabilized by the same non covalent interactions and disulfide bonds as the tertiary structure. omplexes of two or more polypeptides are called multimers. pecifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, a tetramer if it contains four subunits, etc. The subunits are frequently related to one another by symmetry operations, such as a 2 fold axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo " (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero ", for example, a heterotetramer, such as the two alpha and two beta chains of hemoglobin. 9 hydrogen bond ionici bond tyrosine lysine ammonium ions isoleucine alanine valine threonine hydrogen bond 2 histidine Protein dispersion 3 forces phenylalanine carboxylate ions pi stacking phenylalanine leucine dispersion forces cysteine 3 3 alanine dispersion forces 3 2 dispersion forces nonpolar forces are more common on the inside where they can avoid water (hydrophobic) cysteine Forces of interaction (strength). covalent (disulfides) 2. ionic 3. hydrogen bonds 4. dispersion forces 4. pi stacking (similar) methionine arginine valine phosphorylated serine tryptophan 2 arginine 2 P 2 ionic bond polar forces are more common on the outside where they can interact with water (hydrophilic)

11 ne possible way disulfide bonds can form involves cytochrome P-450s and oxygen, (previous topic). There are other possibilities too. Fe +4 cytochrome P-450 sulfur substrate (e-) (nitrogen too) Fe +4 sulfur substrate Fe +3 sulfoxides water sulfoxides disulfides A second way to make disulfide bonds uses lipoamide. lipoamide sulfoxides thiol disulfides FAD 2 FAD 2 Vasopressin has two primary functions in the body: to retain water and to constrict blood vessels. It is synthesized in the hypothalamus and stored in vesicles at the posterior pituitary, where it is released into the bloodstream. It is thought to have an important role in social behavior and has a very short half life between 6 24 minutes. The hormone, oxytocin, is released by the pituitary gland, located in the hypothalamus. The functions of oxytocin include maternal bonding, milk production, uterine contractions during labor, sexual pleasure, reduced fear, and love. 22

12 Insulin has 3 disulfide bonds (lots of post translational modification) GLUT4 containing vesicles fuse to the membrane to allow glucose into the cell. 23 Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. ymptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can cause many complications. Acute complications include diabetic ketoacidosis and nonketotic hyperosmolar coma. erious long-term complications include cardiovascular disease, stroke, chronic kidney failure, foot ulcers, and damage to the eyes. Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. There are three main types of diabetes mellitus: Type DM results from the pancreas's failure to produce enough insulin. This form was previously referred to as "insulin-dependent diabetes mellitus" or "juvenile diabetes". The cause is unknown. Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also develop. This form was previously referred to as "non insulin-dependent diabetes mellitus" or "adult-onset diabetes". The primary cause is excessive body weight and not enough exercise. Gestational diabetes, is the third main form and occurs when pregnant women without a previous history of diabetes develop high blood-sugar levels. It increases the risk of pre-eclampsia p (high blood pressure, protein in urine), depression, and requiring a aesarean section. Prevention is by maintaining a healthy weight and exercising before pregnancy. Gestational diabetes is a treated with a diabetic diet, exercise, and possibly insulin injections. Most women are able to manage their blood sugar with a diet and exercise. reastfeeding is recommended as soon as possible after birth. 24 2

13 Prevention and treatment involve a healthy diet, physical exercise, maintaining a normal body weight, and avoiding use of tobacco. ontrol of blood pressure and maintaining proper foot care are important for people with the disease. Type DM must be managed with insulin injections. Type 2 DM may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar. Weight loss surgery in those with obesity is sometimes an effective measure in those with type 2 DM. Gestational diabetes usually resolves after the birth of the baby. As of 205, an estimated 45 million people have diabetes worldwide, with type 2 DM making up about 90% of the cases. This represents 8.3% of the adult population, with equal rates in both women and men. From 202 to 205, diabetes is estimated to have resulted in.5 to 5.0 million deaths each year. Diabetes at least doubles a person's risk of death. The number of people with diabetes is expected to rise to 592 million by The global economic cost of diabetes in 204 was estimated to be $62 billion UD. In the United tates, diabetes cost $245 billion in The amino acid sequence of a protein determines its native conformation and it folds spontaneously during or after biosynthesis. The process also depends on the solvent (water or lipid bilayer), the concentration of salts, the p, the temperature, the presence of cofactors and of molecular chaperones. Many, many, many decisions (interactions) lead to localized minima, which leads to an overall structure Minimizing the number of hydrophobic side chains exposed to water is an important driving force in protein folding (maximizing entropy of water). Formation of intramolecular hydrogen bonds is another important contribution to protein stability, more so in a hydrophobic core than bonds exposed to the aqueous environment. haperone assisted assisted folding is often necessary in the crowded intracellular environment. Aggregated misfolded proteins are associated with prion related illnesses such as mad cow disease, amyloidrelated illnesses such as Alzheimer's disease, untington's and Parkinson's disease. 26 3

14 ydrophobic forces can be important in quaternary structures. (on the outside) ydrophobic surface faces outside toward lipid bilayer, polar channel on the inside. ydrophobic surface ydrophobic surfaces face towards each other to minimize structuring water molecules Post translational modifications of proteins -acylation protein oa acetyl oa ot protonated at body p when an amide. protein oa hydroxylation collagen collagen collagen collagen proline +4 Fe collagen collagen +4 Fe Fe +3 Makes stronger interactions with neighbors via bonds. hydroxyproline 4% of amino acids in animal tissue. carboxylation protein simplified biotin protein More acidic when acitivated by 2 x =. simplified biotin phosphorylation - turns enzymes on and turns enzymes off. serine, threonine or tyrosine P P ADP protein P P P ATP acyl-like Mg +2 substitution ATP reaction Does the Mg+2 make ADP a better or poorer leaving group? protein P Mg +2 ADP = leaving group an turn on or turn off an enzyme. 28 4

15 Enzymes as receptors and transporters Enzymes life s catalysts eceptors life s communication system tructural Proteins life s framework a+/k+ ATPase (sodium-potassium pump) is an enzyme found in the plasma membrane of all animal cells. The a+/k+ ATPase enzyme is a solute pump that pumps sodium out of cells while pumping potassium into cells, both against their concentration gradients. (it uses energy from ATP). 29 tructural proteins in cell division Microtubules are crucial for cell division. Taxol Paclitaxel is used to treat ovarian, breast, lung, pancreatic and other cancers. Paclitaxel stabilizes the microtubule polymer and stops it from disassembly, preventing cell division. Discovered in 960s in bark of slow growing Yew tree (> 600 years to grow, 3-6 trees = patient, not sustainable). Precursor later discovered in needles or ornamental Yew tree. Even later, genes were spliced into bacteria to synthesize precursor. 30 5

16 Interphase: ells may appear inactive during this stage, but they are quite the opposite. This is the longest period of the complete cell cycle during which DA replicates, the centrioles divide, and proteins are actively produced. Prophase: During this first mitotic stage, the nucleolus fades and chromatin (replicated DA and associated proteins) condenses into chromosomes. Each replicated chromosome comprises two chromatids, both with the same genetic information. Microtubules of the cytoskeleton, responsible for cell shape, motility and attachment to other cells during interphase, disassemble, and the building blocks of these microtubules are used to grow the mitotic spindle from the region of the centrosomes. Prometaphase: In this stage the nuclear envelope breaks down so there is no longer a recognizable nucleus. ome mitotic spindle fibers elongate from the centrosomes and attach to kinetochores, protein bundles at the centromere region on the chromosomes where sister chromatids are joined. ther spindle fibers elongate but instead of attaching to chromosomes, overlap each other at the cell center. Metaphase: Tension applied by the spindle fibers aligns all chromosomes in one plane at the center of the cell. Anaphase: pindle fibers shorten, the kinetochores separate, and the chromatids (daughter chromosomes) are pulled apart and begin moving to the cell poles. Telophase: The daughter chromosomes arrive at the poles and the spindle fibers that have pulled them apart disappear. ytokinesis: The spindle fibers not attached to chromosomes begin breaking down until only that portion of overlap is left. It is in this region that a contractile ring cleaves the cell into two daughter cells. Microtubules then reorganize into a new cytoskeleton for the return to interphase. 3 6