biochem chapter 6 notes

Side Chain Location

AA sidechains in globular proteins are spatially distributed based on their polarities Non-polar residues (V, L, I , M, F) are in the protein interior Out of contact with aqueous environment Charged polar residues (R, H, K, D, E) are located on protein surface Energetically favorable for ion to interact with solvent Uncharged residues (S, T, N, Q, Y) are mostly on surface but can occur inside as well

Amphipathic helices

Amphipathic: contains both polar (hydrophilic) and non-polar (hydrophobic) components Amphipathic helices present discreet areas that are hydrophobic and hydrophilic

Question: What is the difference between a domain and a motif?

Answer: A motif is a chain-like biological structure made up of connectivity between secondary structural pieces, whereas a domain is a three-dimensional protein structure's independent folding unit. Domains are usually larger than motifs, can fold independently, and usually have their own independent function (they will exhibit this even when isolated from the rest of the protein).

Extended Conformation of Polypeptide

Backbone can be drawn as a linked sequence of rigid peptide groups Conformation of the backboneis described by torsion angles(also called dihedral angles):F (phi) Ca-N y (psi) Ca-C Defined as 180o when peptide is fully extended • Increases in clockwise direction Angles constrained by potential steric clashing between side chains, N-H, and C=O

Modified Residues in collagen

Collagen is 1/3 Gly Remaining 2/3 is HyPro Hydroxyproline residues are synthesized by an enzyme (proyl hydroxylase) that uses Vitamin C as a cofactor Pro residues are converted to HyPro after incorporation into the peptide Scurvy results from Vitamin C deficiency (lack of fresh food) Newly synthesized collagen can't form properly Symptoms: loss of teeth, skin lesions, poor wound healing Historically, sailors provided with limes ('limeys")

Collagen: Triple Helix

Collagen is the most abundant protein in vertebrates Strong, insoluble fibers (rope-like) High tensile strength Stress-bearing connective tissue i.e. cartilage, tendons, sub-dermal matrix Each α-chain forms a left-handed helix that twist into a right-handed super helix held together by H-bonds 3 residues per turn every 3rd residue located in the center of the triple helix only enough space for Gly AA sequence of the α-chain is characterized by the Gly-X-Y repeat X and Y often being proline and hydroxyproline residues. NH of Gly H-bonds with C=O of X

Peptide Bonds are planar and rigid

Early x-ray crystal studies revealed the conformational constraints on a polypeptide chain Peptide bonds have a planar, rigid structure due to resonance • 40% double bond character • Maximizes p bond overlap • Evidenced by bond lengths (note differences in C-N bond lengths) • Residues exhibit a dipole

Pleating of β Sheet

In order to avoid steric clashes, beta-strands take on an accordion pleated appearanceSideview shows that all side chain R groups are alternately above or below the plane of the strand, pointing away from the center

NMR Structure of a Protein

Inter-proton distances are imprecise NMR structures are an "ensemble" of closely related structures The precision of structures from multiple measurements suggests an accurate structure Resolution of 2.0-2.5 angstrom Current upper limit for structures is ~100 kDa

Protein Classification: α, β, or α/β

Most proteins are αβ-protein On average ~31% α-helix, ~28% β-sheet slide 40

2o Structure: b-strands form Antiparallel β-Pleated Sheet

The polypeptide backbone in a beta sheet is almost fully extended (rather than being coiled like an a-helix) • Held by inter-strand H-bonding of backbone atoms • Residues alternate facing either side of the main axis • The NH and CO of each amino acid in one b-strand are hydrogen bonded to CO and NH groups of a partner amino acid on another b-strand to form a b-sheet. • Each strand is hydrogen bonded to its neighbors in both directions. • Translation (the distance between adjacent amino acids) = 3.5 Å

Connecting Structures

The residues comprising Type II turns are typically Pro and Gly Pro establishes geometry for H-bonding Gly is small and won't cause steric clashing with C=O of Pro Turns are used for tight reversals of the sequence Secondary structures can be connected by reverse turns (also called β-bends). 4 AA residues long Types differ by inversion of residue 2 C=O

how proteins fold

slide 55 to minimize hydration layer

Scurvy: Vitamin C Deficiency Structure of Collagen Model Peptide

26-27

b-Sheets are not flat

Groups of two to five parallel or antiparallel strands folded together into a b-sheet are common • Inter-strand H-bonds does NOT necessarily imply different polypeptide chains b-sheets are often twisted, with a curved surface• A protein can have a mixture of parallel and antiparallel strands • b-sheets have 2-22 strands in proteins (avg 6); each strand can have up to 15 residues • Connected by simple loops (anti-parallel) or crossovers (parallel)

Helical Wheel diagrams

Helical wheel diagrams give a "top-down" view of the helix Gives visual of residue distribution Residues assigned in order according to this scheme (Note each turn is 3.6 residues):

Metal Ion Stabilized Zinc Finger

Metal ions may also cross-link proteins internally Zinc fingers are 25-60 AA and coordinate around one or 2 Zn2+ ions Component of nucleic acid binding proteins Tetrahedral coordination Typically Cys and His Sometimes Asp or Glu Stabilizes short peptides to permit folding Zn has only one oxidation state so it has predicable bonding geometry that won't change under physiological conditions

Electrostatic forces contribute to protein stability

Non-covalent forces contribute to protein stability, HOWEVER, these are all small contributors compared to the hydrophobic effect: • Electrostatic Interactions (5 kcal/mol)• Hydrogen-bond Interactions (3-7 kcal/mol) • Van Der Waals Interactions (1 kcal/mol)• Hydrophobic Interactions (< 10 kcal/mol) VDW forces are gained when a protein folds due to close contact of interior residues H-bonds contribute -2-8 kj/mol of stability when the protein folds Salt bridges occur when two ionic side chains of opposite charge (e.g. Lys and Asp) associate Usually on protein surface

Ramachandran Diagram for ratphenylalanine hydroxylase

Notice that there are red dots that fall outside the normal range• These are Gly residues with high degrees of freedom • Non-glycine residues occasionally fall outside the norm Pro and residues preceding Pro are omitted for clarity (why?) Ramachandran Diagram for ratphenylalanine hydroxylaseNote that the protein exhibitshigh a-helical content

Symmetries of Oligomeric Proteins

Oligomeric proteins exhibit rotational symmetry Cyclic symmetry (c) is the simplest Single plane with n-fold symmetry Dihedral symmetry is more complicated N-fold symmetry approaching at right angles in a two-fold axis Other forms exist also

Omega Loops

Omega Loops serve a similar function as turns (chain reversal), but their structure is less well-defined, and they are larger. 6 - 16 residue loops often occur at the protein surface Turns and loops often participate in interactions between proteins and other molecules. Often involved in biological recognition processes (for example the antigen-binding sites on Antibodies involve omega loops). Side chains fill most of internal cavity

Peptide groups are usually trans

Peptide bonds assume a trans conformation where successive Ca atomsare on opposite sides of the peptide bond • The trans conformation is 8 kj/molmore stable than cis (Ca on same sideof bond) • Steric interference between clashing groups destabilizes the cisconformation Steric clashing is reduced in prolineresidues • 10% of proline residues are within cispeptide bonds

Structure Conserved More Than Sequence

Polypeptides with similar sequences tend to adopt similar backbone conformations X-ray crystal structures of cytochrome c (eukaryotes) and C-type cytochromes (prokaryotes) are very similar Primary sequences are significantly different Common function (electron carriers) Differ mainly in exterior loops (structures otherwise very similar)

X-Ray Crystallography (I)

Protein crystallization is the process of formation of a regular array of individual protein molecules stabilized by crystal contacts Starts with a pure, highly concentrated protein sample in solution the liquid portion of the solution is slowly evaporated, leaving behind protein crystals. If the crystal is sufficiently ordered, it will diffract light Developing protein crystals is a difficult process and sensitive to pH, temperature, ionic strength in the crystallization solution, and even gravity.

2-Domain Protein : GAPDH

Proteins > 200 AA residues usually fold into 2 or more globular clusters called domains Gives proteins a "lobed" appearance Most domains are 40-200 AA Domains typically have their own independent organization Hydrophobic core/polar surface Domains are often structurally independent and behave as small globular proteins Can fold independently Domains usually have a specific function The Rossmann fold of G3P dehydrogenase binds NAD+ This domain is found in multiple other proteins and exhibits the same function

Methods to Denature Proteins

Proteins are unfolded by disrupting the weak interactions that hold the native structure together Heat- breaks apart non-covalent forces cooperatively, causing the protein structure to "melt" (optical rotation, UV absorbance, and viscosity change drastically) pH variations alter the ionization state of protein side chains changing charge distribution and H-bonding patterns Detergents associate with hydrophobic residues and pull the core apart Chaotropic reagents are ions or small organic molecules that increase the solubility of non-polar substances in water

4º Structure of Hemoglobin

Quaternary protein structure: the organization among multiple peptide chains in a multimeric protein Most multimeric proteins contain an even number of subunits The subunits are held together mainly by hydrophobic interactions between AA sidechain groups noncovalent interactions of tertiary structure also contributes (H-bonding, ionic interactions) Subunits can function independently or cooperatively Easily disrupted by cellular conditions Oligomer: proteins with more than one subunit Protomer: a single subunit of an oligomer

Ramachandran Diagram

Ramachandran plots summarizesterically allowed dihedral angles• A limited # of conformations arefavorable while someconformations are forbidden(VDW radii too close) ieal combinations of f and :• Blue areas: sterically allowed• Green areas are less ideal• Orange circles indicate conformational angles of certain 2o structures a-helixb-sheet, parallelb-sheet, anti-parallel Collagen helixa-helix, left-handed Exceptions:Pro: most rigid (f must be -60o)Gly: most flexible (can adopt "forbidden"dihedral angles)

Sequence Affects 2º Structure

Residues exhibit propensities (likelihood) for being present in certain secondary structures A propensity greater than 1 indicates a "preference" of a secondary structure by that residue Determined by geometric constraints and energy minimization (H-bonding) potential of an AA Useful for predicting secondary structures of proteins with known sequence

The α-Helix

Right-handed "rod-like" structure • Backbone forms the inner part of the rod, theside chains stick outward in a-helical array. 2. The a-helix is stabilized by intra-strand H-bonds between backbone CO and NH groups in the same segment of polypeptide. 3. Multiple H-bonds form between backbone C=O of nth residue and NH of (n + 4)th residue (4 residues ahead in the linear amino acid sequence) 4. H-bond is linear and N...O distance =2.8 Å 5. All the main chain CO and NH groups are H-bonded, which stabilizes these polar groups in the interior of the protein.• Polar groups in the mostly nonpolar interior of a protein need to be involved in favorable interactions, otherwise they would be at thes urface of the protein (opposite charges stabilize) Pro is an a-helix breaker

2-D NMR (COSY)

Solution structures of proteins can be found by NMR Helpful when obtaining a protein crystal proves difficult 1-D NMR is too simple for the # of atoms in a protein COSY is a 2D NMR experiment (homonuclear COrrelation SpectroscopY) that runs decoupling pulses along the entire frequency range Identifies spins between protons which are coupled to each other (usually up to four bonds)

Question: "Hard" keratin (hair, horn, nail) have a higher sulfur content than "soft" keratin (skin and calluses). What do you suppose is the reason?

Sulfur content is proportional to the number of cysteine residues which can make disulfide bonds. Cross-linking strands with disulfide bonds makes keratin resist deformation.

Motifs: Supersecondary Structures

Super secondary structures (or motifs) are groupings of secondary structural elements βαβ- α-helix connect 2 β-strands of a β-sheet β-hairpin- antiparallel sheets with tight β-turns αα- 2 successive antiparallel helices that pack against each other (not a coiled coil, which are parallel) A folded over β-hairpin; 4-stranded antiparallel β-sheet

Advantages of multi-subunit proteins

Synthesis of subunits may be more efficient In supramolecular complexes replacement of worn-out components can be handled more effectively Biological function may be regulated by complex interactions of multiple subunits

NOESY Spectrum of a Protein

The NOESY experiment uses the dipolar interaction of spins (the nuclear Overhauser effect, NOE) for correlation of protons. Intensity of the NOE is proportional to 1/r6, with r being the distance between the protons A signal is only observed if their distance is smaller than 5 Å NOESY correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure

Hydrophobic & Hydrophilic Tendencies: Hydropathy

The hydrophobic effect is the main determinant of native protein structure Recall increase in entropy as solvent becomes more disordered The combined hydrophobic and hydrophilic tendencies of amino acids can be expressed on a hydropathy scale High value means a higher likelihood for a residue to be found in protein's interior

X-Ray Crystallography (II)

The protein crystal is exposed to a concentrated beam of x-rays X-rays wavelength ~1.5 angstrom covalent bond ~1.5 angstrom A diffraction pattern results from the regular repeating atoms of the crystal Visualized by a radiation detector or photographic film X-rays are produced by particle accelerators and synchrotrons Diffraction maxima (darkness of spots) are used to mathematically construct the structure of the protein

Geometry of the α-Helix

The structure repeats itself every 5.4Angstroms along the helix axis,• the a-helix has a pitch of 5.4Angstroms 2. a-helices have 3.6 amino acid residuesper turn,• a helix with 36 amino acids longwould form 10 turns 3. The separation of residues along thehelix axis is 5.4/3.6 or 1.5 Angstroms, ie the alpha-helix has a rise per residue of 1.5 Angstroms 4. The peptide planes are roughly parallel with the helical axis and the dipoles within the helix are aligned (causing amacro dipole)• all C=O groups point in the same direction and all N-H groups point the other way.• N-terminus isd+ while C-terminus isd

β Sheets

There are two types of b-sheets:• antiparallel with linear inter-strand H-bonds (more stable)• parallel with distorted interstrand H-bonds (less stable, bent H-bonds) Difference is relative orientation of adjacent strands• Flat arrows are used to describe the direction of strands in b-sheets

Protein Topology: 8-Stranded β Barrels

Topology- 3-D surface shape determined by how structures are connected The structures below are all 8-stranded β-barrels but exhibit different 3-D structures: slide 41

X-Ray Crystallography (III)

X-rays interact with electrons (not nuclei) to generate electron density maps Since proteins are highly hydrated (40-60% water by volume) the crystals are "jelly-like" Resolution of crystals is lower for proteins compared to the rigid crystals of small molecules Resolution limit of 1.5-3.0 angstrom (limiting factor) Resolution of diketopiperazine as an example: Electron density map isn't enough for accurate structure Trace of backbone allows positions and orientations of sidechains to be deduced Residues can be fit to the map if primary sequence is known (accurate to 0.1 A)

Globular vs. Fibrous Proteins

fibrous Long, parallel polypeptide chains Usually insoluble Little tertiary structure Cross linkages at regular intervals Mostly insoluble Structural role globular Complex tertiary structure Spherical (globular) shape Usually soluble in water May have quaternary structure Metabolic role

Visuals of the α-Helix

slide 12

Ramachandran plot of GFP

slide 20

Keratin: a Coiled Coil

α-keratin is helical but spacing differs from a regular helix 5.1 angstrom pitch (instead of 5.4) Results from closely associated helices called coiled coils Two helices in a left-handed supercoil 310 AA with 7-residue "pseudo repeat" a - b - c - d - e - f - g - a a and d residues are nonpolar and pack against each other to exclude water