structure methods protein function n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Structure Methods; Protein Function PowerPoint Presentation
Download Presentation
Structure Methods; Protein Function

Loading in 2 Seconds...

play fullscreen
1 / 39

Structure Methods; Protein Function - PowerPoint PPT Presentation


  • 144 Views
  • Uploaded on

Structure Methods; Protein Function. Andy Howard Introductory Biochemistry, Fall 2008 9 September 2008. Special Aspects of Tertiary & Quaternary Structure Structural methods Computation X-ray Crystallography NMR Spectroscopy Cryoelectron Microscopy Other experimental techniques

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Structure Methods; Protein Function' - bing


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
structure methods protein function

Structure Methods; Protein Function

Andy HowardIntroductory Biochemistry, Fall 20089 September 2008

Biochemistry: Structure Methods

topics for today
Special Aspects of Tertiary & Quaternary Structure

Structural methods

Computation

X-ray Crystallography

NMR Spectroscopy

Cryoelectron Microscopy

Other experimental techniques

Protein Functions

Post-translational modification

Specific functions

Structural proteins

Enzymes

Electron transport

Storage & transport Proteins

Hormones & receptors

Nucleic-acid binding proteins

Distributions

Topics for today

Biochemistry: Structure Methods

protein topology
Protein Topology
  • Description of the connectivity of segments of secondary structure and how they do or don’t cross over

Biochemistry: Structure Methods

tim barrel
TIM barrel
  • Alternating ,  creates parallel -pleated sheet
  • Bends around as it goes to create barrel

Biochemistry: Structure Methods

domains
Domains
  • Proteins (including single-polypeptide proteins) often contain roughly self-contained domains
  • Domains often separated by linkers
  • Linkers sometimes flexible or extended or both
  • Cf. fig. 6.36 in G&G

Biochemistry: Structure Methods

generalizations about tertiary structure
Generalizations about Tertiary Structure
  • Most globular proteins contain substantial quantities of secondary structure
  • The non-secondary segments are usually short; few knots or twists
  • Most proteins fold into low-energy structures—either the lowest or at least in a significant local minimum of energy
  • Generally the solvent-accessible surface area of a correctly folded protein is small

Biochemistry: Structure Methods

generalizations about quaternary structure
Generalizations about quaternary structure
  • Considerable symmetry in many quaternary structure patterns (see G&G section 6.5)
  • Weak polar and solvent-exclusion forces add up to provide driving force for association
  • Many quaternary structures are necessary to function: monomer can’t do it on its own in a lot of cases

Biochemistry: Structure Methods

how do we visualize protein structures
How do we visualize protein structures?
  • It’s often as important to decide what to omit as it is to decide what to include
  • Any segment larger than about 10Å needs to be simplified if you want to understand it
  • What you omit depends on what you want to emphasize

Biochemistry: Structure Methods

styles of protein depiction
Styles of protein depiction
  • All atoms
  • All non-H atoms
  • Main-chain (backbone) only
  • One dot per residue (typically at C)
  • Ribbon diagrams:
    • Helical ribbon for helix
    • Flat ribbon for strand
    • Thin string for coil

Biochemistry: Structure Methods

how do we show 3 d
How do we show 3-D?
  • Stereo pairs
    • Rely on the way the brain processes left- and right-eye images
    • If we allow our eyes to go slightly wall-eyed or crossed, the image appears three-dimensional
  • Dynamics: rotation of flat image
  • Perspective (hooray, Renaissance)

Biochemistry: Structure Methods

a more pedestrian application
A more pedestrian application
  • Sso7d bound to DNAGao et al (1998) NSB 5: 782

Biochemistry: Structure Methods

a little more complex
A little more complex:
  • Aligning Cytochrome C5with Cytochrome C550

Biochemistry: Structure Methods

ribbon diagrams
Mostly helical:E.coli RecG - DNA

PDB 1gm53.24Å, 105 kDa

Mixed:hen egg-white lysozyme

PDB 2vb10.65Å, 14.2kDa

Ribbon diagrams

Biochemistry: Structure Methods

warning specialty content
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.

Biochemistry: Structure Methods

how do we determine structure
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

Biochemistry: Structure Methods

crystallography overview
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

Biochemistry: Structure Methods

how are protein crystals unusual
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

Biochemistry: Structure Methods

crystal structures fourier transforms of diffraction results
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(2ik•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) hklFhkl exp(-2ih•r)

Biochemistry: Structure Methods

the phase problem
The phase problem

Fhkl

bhkl

  • Note that we saidIhkl = C*|Fhkl|2
  • That means we can figure out|Fhkl| = (1/C)√Ihkl
  • But we can’t figure out the direction ofF:Fhkl=ahkl + ibhkl = |Fhkl|exp(ihkl)
  • 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!

ahkl

Biochemistry: Structure Methods

what can we learn
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 non-H atoms, most solvent, some disordered atoms; some H’s

Biochemistry: Structure Methods

what does this look like
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

Biochemistry: Structure Methods

how s the field changing
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
    • Most structures will be determined by cell biologists & molecular biologists

Biochemistry: Structure Methods

macromolecular nmr
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

Biochemistry: Structure Methods

typical protein 2 d spectrum
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

Biochemistry: Structure Methods

results
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
  • The ambiguities partly represent real molecular diversity; but they also represent atoms that area in truth well-defined, but the NMR data don’t provide the unambiguous assignment

Biochemistry: Structure Methods

comparing nmr to x ray
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 in NMR;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 > 35 kDa

Biochemistry: Structure Methods

what does it mean when nmr and x ray structures differ
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

Biochemistry: Structure Methods

cryoelectron microscopy
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

Biochemistry: Structure Methods

circular dichroism
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

Biochemistry: Structure Methods

poll question discuss

Sperm whale myoglobinPDB 2jho1.4Å16.9 kDa

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

Anti-fluorescein FabPDB 1flr1.85 Å52 KDa

Biochemistry: Structure Methods

ultraviolet spectroscopy
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

Biochemistry: Structure Methods

protein function generalities
Protein Function: Generalities
  • Proteins do a lot of different things. Why?
  • Well, they’re coded for by the ribosomal factories
  • … But that just backs us up to the question of why the ribosomal mechanism codes for proteins and not something else!

Biochemistry: Structure Methods

proteins are chemically nimble
Proteins are chemically nimble
  • The chemistry of proteins is flexible
    • Protein side chains can participate in many interesting reactions
    • Even main-chain atoms can play roles in certain circumstances.
  • Wide range of hydrophobicity available (from highly water-hating to highly water-loving) within and around proteins gives them versatility that a more unambiguously hydrophilic species (like RNA) or a distinctly hydrophobic species (like a triglyceride) would not be able to acquire.

Biochemistry: Structure Methods

what proteins can do
What proteins can do
  • Proteins can act as catalysts, transporters, scaffolds, signals, or fuel in watery or greasy environments, and can move back and forth between hydrophilic and hydrophobic situations.
  • Furthermore, proteins can operate either in solution, where their locations are undefined within a cell, or anchored to a membrane.
    • Membrane binding keeps them in place.
    • Function may occur within membrane or in an aqueous medium adjacent to the membrane

Biochemistry: Structure Methods

structure function relationships
Structure-function relationships
  • Proteins with known function: structure can tell is how it does its job
    • Example: yeast alcohol dehydrogenase
    • Catalyzesethanol + NAD+ acetaldehyde + NADH + H+
    • We can say something general about the protein and the reaction it catalyzes without knowing anything about its structure
    • But a structural understanding should help us elucidate its catalytic mechanism
  • Protein with unknown function: structure might tell us what the function is!

Biochemistry: Structure Methods

why this example
Why this example?
  • Structures of ADH from several eukaryotic and prokaryotic organisms already known
  • Yeast ADH is clearly important and heavily studied, but until 2006: no structure!
  • We got crystals 7 years ago, but so far I haven’t been able to determine the structure

Yeast ADH

PDB 2hcy2.44Å

152 kDa tetramerdimer shown

Biochemistry: Structure Methods

what we know about this enzyme
What we know about this enzyme
  • Cell contains an enzyme that interconverts ethanol and acetaldehyde, using NAD as the oxidizing agent (or NADH as the reducing agent)
  • We can call it alcohol dehydrogenase or acetaldehyde reductase; in this instance the former name is more common, but that’s fairly arbitrary (contrast with DHFR)

Biochemistry: Structure Methods

size and composition
Size and composition
  • Tetramer of identical polypeptides
  • Total molecular mass = 152 kDa
  • We can do arithmetic: the individual polypeptides have a molecular mass of 38 kDa (347 aa).
  • Human is a bit bigger: 374 aa per subunit
  • Each subunit has an NAD-binding Rossmann fold over part of its structure

Biochemistry: Structure Methods