Protein Structure and Function Andrew Howard Fourth Lecture Introductory Biochemistry 31 January 2008

Protein Structure and Function

Andrew HowardFourth LectureIntroductory Biochemistry

31 January 2008

IIT Biochemistry: 31 Jan 2008 Slide 2 of 60

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.


Let’s finish up peptides, though! We need to talk about Ramachandran angles, peptide bonds, and oligopeptide and polypeptide chemistry before we go on to techniques for determining structures.


Ramachandran angles

G.N. Ramachandran


Preferred Values of and

Steric hindrance makes some values unlikely

Specific values are characteristic of particular types of secondary structure

Most structures with forbidden values of and turn out to be errors


How far from 180º can vary? Remember what we said about the partial double bond character of the C-N main-chain bond

That imposes planarity In practice it rarely varies by more than a few degrees from 180º.


Ramachandran plot Cf. fig. 4.9 in Horton

If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them!


How are oligo- and polypeptides synthesized? Formation of the peptide linkages occurs in the ribosome under careful enzymatic control

Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine):

GTP + n-length-peptide + amino acid GDP + Pi + (n+1)-length peptide


What happens at the ends? Usually there’s a free amino end and a free carboxyl end:

H3N+-CHR-CO-(peptide)n-NH-COO-

Cyclic peptides do occur Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step.


Reactivity in peptides & proteins

Main-chain acid-base reactivity unavailable except on the ends

Side-chain reactivity available but with slightly modified pKas.

Terminal main-chain pKavalues modified too

Environment of protein side chain is often hydrophobic, unlike free amino acid side chain’s neighborhood


Discussion questionWhat’s the net charge in ELVISat pH 7?

(a) 0 (b) +1 (c) -1 (d) +2 (e) -2


Disulfides

In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

CHHSHCHHSH+(1/2)O2SSHCHHCHH2O


What could this do? Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another

This can influence protein stability


… and now, onward to protein structure methods and results We’ll look at several techniques, and then focus on what we know on the basis of the application of those techniques.


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.


How do we determine structure? We can distinguish between methods that require little prior knowledge (crystallography, NMR, ?CryoEM?)and methods that answer specific questions (XAFS, fiber, …)

This distinction isn’t entirely clear-cut


Crystallography: overview Crystals are translationally ordered 3-D arrays of molecules

Conventional solids are usually crystals

Proteins have to be coerced into crystallizing

… but once they’re crystals, they behave like other crystals, mostly


How are protein crystals unusual? Aqueous interactions required for crystal integrity: they disintegrate if dried

Bigger unit cells (~10nm, not 1nm) Small # of unit cells and static disorder means they don’t scatter terribly well

So using them to determine 3D structures is feasible but difficult


Crystal 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

Documents

Protein Structure and Function Andrew Howard Fourth Lecture Introductory Biochemistry 31 January 2008