09/04/08Biochemistry: Methods & Structure Protein Methods; Fundamentals of Protein Structure Andy...

09/04/08Biochemistry: Methods & Structure

Protein Methods;Fundamentals of Protein

Structure

Andy HowardIntroductory Biochemistry, Fall 2008

4 September 2008

09/04/08Biochemistry: Methods & Structure of 38

Plans for Today Protein methods

(Concluded) Electrophoresis Spectroscopy Scattering

Why we care about structure

Levels of Structure Primary Secondary Tertiary Quaternary


Electrophoresis Separating analytes by charge by

subjecting a mixture to a strong electric field

Gel electrophoresis: field applied to a semisolid matrix

Can be used for charge (directly) or size (indirectly)


SDS-PAGE Sodium dodecyl sulfate: strong

detergent, applied to protein Charged species binds quantitatively Denatures protein

Good because initial shape irrelevant Bad because it’s no longer folded

Larger proteins move slower because they get tangled in the matrix

1/Velocity √MW


SDS PAGE illustrated


Isoelectric focusing Protein applied to gel without

charged denaturant Electric field set up over a pH

gradient (typically pH 2 to 12) Protein will travel until it reaches

the pH wherecharge =0 (isoelectric point)

Sensitive to single changes in charge (e.g. asp -> asn)

Readily used preparatively with samples that are already semi-pure


Ultraviolet spectroscopy Tyr, trp absorb and fluoresce:

abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr)

Reliable enough to use for estimating protein concentration via Beer’s law

UV absorption peaks for cofactors in various states are well-understood

More relevant for identification of moieties than for structure determination

Quenching of fluorescence sometimes provides structural information


X-ray spectroscopy

All atoms absorb UV or X-rays at characteristic wavelengths

Higher Z means higher energy, lower for a particular edge

Perturbation of absorption spectra at E = Epeak + yields neighbor information

Changes just below the peak yield oxidation-state information

X-ray relevant for metals, Se, I


Solution scattering Proteins in solution scatter X-rays

in characteristic, spherically-averaged ways

Low-resolution structural information available

Does not require crystals Until ~ 2000 you needed high

[protein] Thanks to BioCAT, SAXS on dilute

proteins is becoming more feasible Hypothesis-based analysis


Fiber Diffraction

Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering)

Produce characteristic fiber diffraction patterns

Collagen, muscle proteins, filamentous viruses


Protein Structure Helps us Understand Protein Function If we do know what a protein does,

its structure will tell us how it does it. If we don’t know what a protein

does, its structure might give us what we need to know to figure out its function.


Levels of Protein Structure We conventionally describe proteins at

four levels of structure, from most local to most global: Primary: linear sequence of peptide units and

covalent disulfide bonds Secondary: main-chain H-bonds that define

short-range order in structure Tertiary: three-dimensional fold of a

polypeptide Quaternary: Folds of multiple polypeptide

chains to form a complete oligomeric unit


What does the primary structure look like? -ala-glu-val-thr-asp-pro-gly- … Can be determined by amino acid sequencing of

the protein Can also be determined by sequencing the gene

and then using the codon information to define the protein sequence

Amino acid analysis means percentages; that’s less informative than the sequence


Components of secondary structure

, 310, helices pleated sheets and

the strands that comprise them

Beta turns More specialized

structures like collagen helices


An accounting for secondary structure: phospholipase A2


Alpha helix


Characteristics of helices

Hydrogen bonding from amino nitrogen to carbonyl oxygen in the residue 4 earlier in the chain

3.6 residues per turn Amino acid side chains face outward ~ 10 residues long in globular proteins


What would disrupt this? Not much: the side chains

don’t bump into one another Proline residue will disrupt it:

Main-chain N can’t H-bond The ring forces a kink

Glycines sometimes disrupt because they tend to be flexible


Other helices

NH to C=O four residues earlier is not the only pattern found in proteins

310 helix is NH to C=O three residues earlier More kinked; 3 residues per turn Often one H-bond of this kind at N-

terminal end of an otherwise -helix helix: even rarer: NH to C=O

five residues earlier


Beta strands

Structures containing roughly extended polypeptide strands

Extended conformation stabilized by inter-strand main-chain hydrogen bonds

No defined interval in sequence number between amino acids involved in H-bond


Sheets: roughly planar Folds straighten H-bonds Side-chains roughly

perpendicular from sheet plane

Consecutive side chains up, then down

Minimizes intra-chain collisions between bulky side chains


Anti-parallel beta sheet

Neighboring strands extend in opposite directions

Complementary C=O…N bonds from top to bottom and bottom to top strand

Slightly pleated for optimal H-bond strength


Parallel Beta Sheet

N-to-C directions are the same for both strands

You need to get from the C-end of one strand to the N-end of the other strand somehow

H-bonds at more of an angle relative to the approximate strand directions

Therefore: more pleated than anti-parallel sheet


Beta turns Abrupt change in direction , angles are

characteristic of beta Main-chain H-bonds

maintained almost all the way through the turn

Jane Richardson and others have characterized several types


Collagen triple helix

Three left-handed helical strands interwoven with a specific hydrogen-bonding interaction

Every 3rd residue approaches other strands closely: so they’re glycines


Poll question Remember that there are

about 3.6 residues per turn in an alpha helix.

Suppose you had a helical protein that was sitting on, not in, a phospholipid bilayer so that the side chains point inward and outward along the surface.

Which of the following sequences would be the most stable in this environment?


Options Assume side chain of

residue 2 points DOWN into the bilayer: (a) GADHKYEKLRG (b) GLDGIVESVGG (c) AKRTTVWKDKD (d) YRNNADRRKLG


Note about disulfides

Cysteine residues brought into proximity under oxidizing conditions can form a disulfide

Forms a “cystine” residue Oxygen isn’t always the

oxidizing agent Can bring sequence-distant

residues close together and stabilize the protein

CHHSHCHHSH+(1/2)O2SSHCHHCHH2O


Hydrogen bonds, revisited Biological settings, H-bonds are almost

always: Between carbonyl oxygen and hydroxyl:

(C=O ••• H-O-) between carbonyl oxygen and amine:

(C=O ••• H-N-) These are stabilizing structures

Any stabilization is (on its own) entropically disfavored;

Sufficient enthalpic optimization overcomes that!

In general the optimization is ~ 1- 4 kcal/mol


Secondary structures in structural proteins

Structural proteins often have uniform secondary structures

Seeing instances of secondary structure provides a path toward understanding them in globular proteins

Examples: Alpha-keratin (hair, wool, nails, …): -helical Silk fibroin (guess) is -sheet


Alpha-keratin Actual -keratins

sometimes contain helical globular domains surrounding a fibrous domain

Fibrous domain: long segments of regular -helical bonding patterns

Side chains stick out from the axis of the helix


Silk fibroin

Antiparallel beta sheets running parallel to the silk fiber axis

Multiple repeats of (Gly-Ser-Gly-Ala-Gly-Ala)n


Secondary structure in globular proteins Segments with secondary structure are usually

short: 2-30 residues Some globular proteins are almost all helical,

but even then there are bends between short helices

Other proteins: mostly beta Others: regular alternation of , Still others: irregular , , “coil”


Tertiary Structure

The overall 3-D arrangement of atoms in a single polypeptide chain

Made up of secondary-structure elements & locally unstructured strands

Described in terms of sequence, topology, overall fold, domains

Stabilized by van der Waals interactions, hydrogen bonds, disulfides, . . .


Quaternary structure

Arrangement of individual polypeptide chains to form a complete oligomeric, functional protein

Individual chains can be identical or different If they’re the same, they can be coded for

by the same gene If they’re different, you need more than

one gene


Not all proteins have all four levels of structure Monomeric proteins don’t have

quaternary structure Tertiary structure: subsumed into

2ndry structure for many structural proteins (keratin, silk fibroin, …)

Some proteins (usually small ones) have no definite secondary or tertiary structure; they flop around!


Protein Topology Description of the

connectivity of segments of secondary structure and how they do or don’t cross over


TIM barrel Alternating , creates parallel -

pleated sheet Bends around as it goes to create

barrel

09/04/08Biochemistry: Methods & Structure Protein Methods; Fundamentals of Protein Structure Andy...

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