These are notes from lecture 2 of Harvard Extension’s biochemistry class.
The bicarbonate system maintains blood pH.
lung CO2 ↔ blood CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3-
So when you get too much CO2 your blood pH drops, triggering a “blow off” CO2 response.
This class’s particular classification of amino acids:
|nonpolar||G, A, P, V, F, W, L, I, M|
|polar uncharged||S, T, C, Y, N, Q|
|positively charged*||K, R, H**|
|negatively charged*||D, E|
*At physiological pH
**Histidine’s pKa is very close to physiological pH, so it is sometimes protonated (and therefore positively charged) and sometimes not
Each amino acid has a particular pKa for each of its -COOH and -NH3+; many also have a pKa for their R group. pKa of COOH is usually ~2 and of NH3+ is usually ~9.5. See this table of the pKas.
In acid-base titration, amino acids will hit a wall where all the NH3+ is protonated and all the COO- is deprotonated, thus it is net neutral. This is its isoelectric point or pI: the pH at which the net charge is zero. ”This” is called the Zwitterion. ?? At pH > pI, an AA is negatively charged; at pH < pI, it is positively charged.
For AAs without an ionizable R group, the pI is ½(pKC + pKN). For those with an ionizable group and pKR < pKN, pI = ½*(pKC + pKR); if pKR > pKN, pI = ½*(pKN + pKR). (Note: she uses pK1 to mean pKC and pK2 to mean pKN).
Proteins of < 40 aa are called “oligopeptides”. Those of MW < 10 kDa are “polypeptides” and > 10 kDa are “proteins”. Average amino acid molecular weight is ~110 Da, thus 10 kDa ~= 90 aa.
Peptide bonds have a resonance structure with O-, NH+ and a double bond C=N. Thus there is some double bond character in a peptide bond, which prevents rotation around the C=N bond. The C in COOH is called Cα; the C in the center of the tetrahedron is just called C. Ψ (Psi) is rotation around the C-αC bond. Φ (Phi) is rotation around the N-Cα bond.
Secondary structure requirements:
- Must allow the polar peptide bond to hydrogen bond: O- to be a hydrogen bond acceptor, and NH+ to be a hydrogen bond donor.
- Minimize steric strain.
- Position side chains to minimize interference. (Isn’t this also steric??)
Two secondary structures meet these well: α-helices and β-sheets
α-helices are a spiral staircase where you walk up the staircase with your right hand on the outer railing and your left hand on the central pole. Rungs are 5.4 Å (3.6 aa) high to satisfy hydrogen bonding between one aa’s O- and another’s N+. Side chains point outward from the helix. R groups of amino acids affect their ability to participate in an α-helix. Because P has its R group covalently bonded to NH2+, it forces a kink in the chain which makes it very rare in helices. G, meanwhile, is too flexible and is therefore also rare in α-helices. Also too many negatives (e.g. EEEEE) or too many positives (e.g. KKKKK) repel each other and also cannot form a helix. Too many bulk proteins (N, T, Q) is also destabilizing.
β-sheets can be parallel (N→C, N→C, N→C) or antiparallel (N→C, C→N, N→C). The backbone hydrogen bonds are in between the strands. R groups within the sheets need to be small – G and A are common. There are on average 6 strands per β-sheet.
Irregular secondary structures called loops and β-turns link multiple helices or sheets. Loops link antiparallel sheets, β-turns link parallel sheets.
Globular proteins have a hydrophilic surface and a hydrophobic core. The core is rich in hydrophobic amino acids; any polar residues present there must hydrogen-bond to neutralize them, and charged residues must participate in ionic bonding. The core is rich in regular secondary structure – the internal hydrogen bonding of helices and sheets minimizes the chargedness. The surface is enriched for polar and charged amino acids.
A motif is a supersecondary structure – a pattern of ordering of sheets and helices. e.g. Greek key.
A domain is a part of a peptide chain that is independently stable or can move as a single entity with respect to the rest of the protein. The strict definition (?) is that if you separate 2 domains, each will retain its own individual structure.
Homo-oligomers (dimers, trimers, tetramers) have all subunits identical; hetero-oligomers have non-identical subunits.
A protein will fold into the “most thermodynamically stable” state (lowest Gibbs free energy). The hydrophobic effect is the main driving force that drives protein folding. A disordered (unfolded) peptide means ordered “caged water”. Net entropy is actually increased by folding the protein and thus freeing the water from its cage.
Secondary to the hydrophobic effect, other thermodynamic forces also contribute to protein folding:
- Van der Waals, especially in the hydrophobic core of a globular protein
- hydrogen bonding of R groups, though these don’t add much to the driving force because they can just as easily bond with water as with each other
- ionic interactions or “salt bridges” between charged R groups.
- covalent disulfide bonds, though these are primarily (only?) found in extracellular proteins (her explanation: the environment is harsher so the covalent bonds are needed to support the protein structure).
Anifinsen (Nobel 1972) hypothesized that primary structure drives secondary and tertiary structure. To test this he asked whether a protein denatured with urea and reduced (eliminating disulfide bonds) with beta mercaptoethanol will re-fold to its native conformation. In the particular protein he studied, ribonuclease A, the answer happened to be yes.
It is hypothesized that spontaneous protein folding involves 2 events: (1) almost-instant folding of local segments of secondary structure, and (2) hydrophobic collapse in a “molten globule.”
The thermodynamics of folding are sometimes described as a “funnel”. As you fold, energy decreases but entropy also decreases, with many intermediates that are a local minimum but ultimately break and re-fold, until you get to the bottom of the well which is the “native structure.”
In reality many proteins require chaperones. One example is Hsp70 holding a protein unfolded until it can be transferred to Hsp60 which is visualized as a “cage” inside which the protein can fold.
In the “molten globule” stage between denatured and native states, a β-sheet rich protein can oligomerize to form amyloid fibrils.
O2 is poorly soluble in aqueous solution and cannot diffuse more than a few mm. Therefore we evolved proteins to carry it.
Myoglobin has a “globin fold motif”. It is a polypeptide with eight &alpha-helices and one heme group. It can store O2 (a prominent role in cetaceans) or faciliate O2 diffusion in muscle (a prominent role in land mammals). Heme is a porphyrin ring with four nitrogens coordinating one Fe2+ which binds O2. It can only bind if the heme is exposed, not if the heme is buried in the protein. Myoglobin binding to O2 follows a hyperbolic curve with partial pressure of oxygen on the x axis. At 2.8 torr, 50% of myoglobin is oxygen-bound – this is called a p50 (by analogy to EC50 in pharmcology?) The lower the p50, the greater the affinity of myoglobin for O2.
Hemoglobin (Hb) is a heterotetramer made of 2 α globin subunits and 2 β globin subunits, each of which has one heme. Hemoglobin has a much higher p50 than myoglobin. Hemoglobin gets saturated with oxygen in the lungs at 100 torr, then as the partial pressure of oxygen drops as you get further from the lungs, it sheds O2. Thus the lower affinity of hemoglobin than myoglobin is crucial for the former’s biological role of delivering oxygen.
Hemoglobin binding to oxygen actually follows a sigmoidal curve – it is “reluctant” to bind its first O2 but once it does, that causes conformational change from a “tense” (T) state to a “relaxed” (R) state (in which the porphyrin is more planar) that increases affinity for additional O2. It binds its 4th O2 with 100x the affinity with which it binds its 1st. This is called “cooperative binding”. Naturally, if there’s cooperative binding there’s also cooperative release – once some oxygen is released, the rest is more prone to release. Hemoglobin’s lower affinity (compared to myoglobin) and cooperative binding are both critical to its biological function.
Hb can also transport H+ and CO2. Only in the T state, it can bind H+ to D94 & H146, and CO2 can bind to the N-terminal group of each globin chain. These interactions stabilize the T state, and so the binding of H+ and CO2 is inversely proportional to O2. High CO2 increases H+ due to equilibrium with dissociated carbonic acid; therefore in tissues with high CO2 and low pH, Hb loses affinity for oxygen and releases it (perfect since those are the tissues that need oxygen). pH’s effect on Hb binding to oxygen is called the Bohr effect.
Effects of 2,3-Bisphopshogylcerate (BPG) on Hb. BPG is constitutively present in red blood cells but increases in response to altitude (low pO2). Without BPG, the T state is unstable and the R state is more favored, thus Hb’s affinity for oxygen would be higher. BPG is present in red blood cells at approximately the same concentration as Hb (1:1 stoichiometry?). 1 BPG molecule binds each Hb molecule in a pocket at the center of the tetramer – a pocket present only in the T state. In the presence of BPG, it takes more O2 binding events to induce the T → R transition.
At sea level, Hb releases 38% of the maximum oxygen it can carry. At altitude it would release only 30% if it had the same affinity, because the partial pressure of oxygen in the lungs is lower. However at altitude, BPG is upregulated, thus stabilizing the T state and reducing the affinity of Hb for oxygen. The reduction in affinity allows it to shed more oxygen by the time it reaches the pO2 found in tissue, for a total of 37% of maximum – approximately the same amount as at sea level.
Micelles. According to Haynes, adding 1 molecule of fatty acid decreases entropy of water, while adding 100 increases entropy b/c of micelle. This argument sounds inconsistent: the driving force for micelle formation has to be the *initially* lower entropy upon adding the 100 molecules.
Today 7:40-8:40 SC 103b – Roopali section