IIT Biochemistry: 17 Sep 2007 Slide 1 of 56 Protein Structure Helps us Understand Protein Function...

IIT Biochemistry: 17 Sep 2007 Slide 1 of 56

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.


Plans for Today

Methods of Determining Protein Structure Crystallography

NMR CryoEM Specialty techniques

Levels of Protein Structure

Hydrogen Bonds Secondary structure in globular proteins

Tertiary Structure

Domains


Warning: Specialty Content! I determine protein structures (and develop methods for determining protein structures) as my own research focus

So it’s hard for me to avoid putting a lot of emphasis on this material

But today I’m allowed to do that, because it’s the stated topic of the day.


Structures: Fourier transforms of diffraction results

Position of spots tells you how big the unit cell is

Intensity tells you what the contents are We’re using electromagnetic radiation, which behaves like a wave, exp(2ik•x)

Therefore intensity Ihkl = C*|Fhkl|2

Fhkl is a complex coefficient in the Fourier transform of the electron density in the unit cell:(r) = (1/V) hkl Fhkl exp(-2ih•r)


The phase problem Note that we said Ihkl = C*|Fhkl|2

That means we can figure out|Fhkl| = (1/C)√Ihkl

But we can’t figure out the direction of F:Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl)

This direction angle is called a phase angle

Because we can’t get it from Ihkl, we have a problem: it’s the phase problem!

F

ab


What can we learn Electron density map + sequence we can determine the positions of all the non-H atoms in the protein—maybe!

Best resolution possible: Dmin = / 2 Often the crystal doesn’t diffract that well, so Dmin is larger—1.5Å, 2.5Å, worse

Dmin ~ 2.5Å tells us where backbone and most side-chain atoms are

Dmin ~ 1.2Å: all protein atoms, most solvent, some disordered atoms


What does this look like? Takes some experience to interpret

Automated fitting programs work pretty well with Dmin < 2.1Å

ATP binding to a protein of unknown function: S.H.Kim


How’s the field changing?

1990: all structures done by professionals

Now: many biochemists and molecular biologists are launching their own structure projects as part of broader functional studies

Fearless prediction: by 2020, crystallographers will be either technicians or methods developers


Macromolecular NMR NMR is a mature field Depends on resonant interaction between EM fields and unpaired nucleons (1H, 15N, 31S)

Raw data yield interatomic distances Conventional spectra of proteins are too muddy to interpret

Multi-dimensional (2-4D) techniques:initial resonances coupled with additional ones


Typical protein 2-D spectrum

Challenge: identify whichH-H distance is responsible for a particular peak

Enormous amount of hypothesis testing required

Prof. Mark Searle,University of Nottingham


Results

Often there’s a family of structures that satisfy the NMR data equally well

Can be portrayed as a series of threads tied down at unambiguous assignments

They portray the protein’s structure in solution


Comparing NMR to X-ray NMR family of structures often reflects real

conformational heterogeneity Nonetheless, it’s hard to visualize what’s

happening at the active site at any instant Hydrogens sometimes well-located;

they’re often the least defined atoms in an X-ray structure

The NMR structure is obtained in solution! Hard to make NMR work if MW > 25 kDa


What does it mean when NMR and X-ray structures differ?

Lattice forces may have tied down or moved surface amino acids in X-ray structure

NMR may have errors in it X-ray may have errors in it (measurable)

X-ray structure often closer to true atomic resolution

X-ray structure has built-in reliability checks


Cryoelectron microscopy

Like X-ray crystallography,EM damages the samples

Samples analyzed < 100Ksurvive better

2-D arrays of molecules Spatial averaging to improve resolution

Discerning details ~ 4Å resolution

Can be used with crystallography


Circular dichroism

Proteins in solution can rotate polarized light

Amount of rotation varies with

Effect depends on interaction with secondary structure elements, esp.

Presence of characteristic patterns in presence of other stuff enables estimate of helical content


Poll question: discuss! Which protein would yield a more interpretable CD spectrum? (a) myoglobin (b) Fab fragment of immunoglobulin G

(c) both would be fully interpretable

(d) CD wouldn’t tell us anything about either protein


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


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


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


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 arecharacteristic 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


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!


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

IIT Biochemistry: 17 Sep 2007 Slide 1 of 56 Protein Structure Helps us Understand Protein Function...

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Transcript of IIT Biochemistry: 17 Sep 2007 Slide 1 of 56 Protein Structure Helps us Understand Protein Function...