Lecture 9 M230 Feigon Sequential resonance assignments in (small) proteins: homonuclear method 2º structure determination Reading resources v Roberts NMR of Macromolecules, Chap 4 by Christina Redfield Evans Chap 4, pp 147-169 Wuthrich NMR of proteins and nucleic acids Wagner & Wuthrich, JMB, 155, 347 (1982)
Sequential resonance assignments in proteins NOESY: info on interproton distances COSY: info on coupling constants, & therefore dihedral angles Before you can use this information to determine structures, you must first assign the resonances. (1) Identification of the aa spin systems using coherence transfer experiments (COSY, TOCSY, many others) à coupling between amide, CH α, CH β (2) Identification of neighbors in the primary sequence à connectivities via NOESY Homonuclear ( 1 H) methods only work for proteins 15 kd; Larger proteins require heteronuclear 3D methods
(D) (D) (D) (D) (1a) Identify non-labile spin systems via COSY, TOCSY, etc of as many aa s as possible Examples: (COSY in D 2 O) (1b) Correlate spin systems of non-labile aa protons with their labile amide proton via COSY in H 2 O or TOCSY in H 2 O NH - C α H (amide - alpha xpeak) ~10.6-6.6 ppm ~6-2 ppm called FINGERPRINT REGION because 1 xpeak/aa except gly (1 or 2); pro (no NH); Nterm or unfolded (may exchange too fast)
Identification of aa spin systems via COSY & TOCSY COSY: TOCSY:,, AA spin systems 6 unique: gly, ala, val, ile, leu, thr 8 AMX: ser, cys, asp, asn; phe, tyr, his, trp [arom] 6 long side chain: gln, glu, met; arg, pro; lys Aromatics also have separate ring systems
3) 1D 1 H Protein spectrum in H 2 O Rnt1p dsrbd ~90 residues 800 MHz H 2 O -CH 2 - -CH 3 Backbone amide NH Plus sidechains resonances from: Trp, Phe, Tyr, His ring H Asn & Gln amides Arg NH Lys NH 2 CHα
Protease inhibitor K COSY in D 2 O *Freshly dissolved in D 2 O, so some slowly exchanging amides still present
From Wagner & Wüthrich, J Mol Biol, 155, 347-366 (1982) Fingerprint region COSY in H 2 O intraresidue (αn ii )
Identification of aa neighbors in 1º sequence For any given aa conformation, a sequential NOE can be observed between one or more of the following: alpha i -amide i+1 amide i -amide i+1 beta i -amide i+1 d αn (i,i+1) = d αn d NN (i,i+1) = d NN d βn (i,i+1) = d βn Note: In general, we know the 1º sequence of the protein. We need to assign the resonances in the spectrum and compare to 1º sequence.
NOESY spectrum of BPTI illustrating the regions of the spectrum used for sequential assignments (1º structure). Other regions in NOESY give info on side chain interactions (2º & 3º structure) Wagner & Wuthrich, J. Mol. Biol. 155, 347 (1982)
To get sequential assignments, compare NOESY xpeaks to amides (αn ij, NN ij, βn ij, αn ii ) with COSY xpeaks to amides (αn ii ); àtherefore can connect individual aas assigned via COSY in 1 sequence (a) αn connectivities In COSY, will have αn ii xpeaks (1/aa residue) In NOESY, will have αn ii and αn i,i+1 xpeaks, but NOT Nα i,i+1 Compare COSY fingerprint region with same region in NOESY. NOESY will have: some xpeaks αn ii same as COSY, plus additional xpeaks αn ij (αn i,i+1 plus others). If there is a xpeak in NOESY at same amide position but different α position as COSY, it is a xpeak between aas i+1 & i (or same α, dif amide, i,i+1) --K 9 A 10 K 11 --
(b) NN connectivities Again, amides are assigned to aa type from COSY, TOCSY, etc. Amides are sequentially connected via NOESY, but NN connectivities are not directional. Example crosspeak patterns for tripeptide (i-1, i, i+1)
Now, combine info from NN, αn, & βn regions. Proteins will usually have several unique dipeptides, many unique tripeptides, and most tetrapeptides are unique. àso, can get assignments even when exact aa not assigned (ie fill in the blanks). Can then interatively go back to spectra to get more assignments. Info combined to get 1 structure of BPTI
Secondary structure determination by NMR The NOEs observed in determining sequential assignment also give information on 2º structure and therefore on folding of proteins. àget specific patterns of NOES for regular 2º structures. (1) β-sheet Easiest 2º structure to characterize Typical distance d αn(i,i+1) ~2.2Å strong NOE d αn(i,i) ~2.8Å med-strong NOE d NN(i,i+1) ~4.2Å weak NOE d βn(i,i+1) ~3.2-4.4Å depends on side chain Therefore, in fingerprint region of NOESY, will see strong xpeaks for NOE to neighbor (i,i+1) but weaker xpeaks where COSY xpeaks were (i,i). àβ-sheet is characterized by strong xpeaks between neighboring aas (αn i,i+1 ) and somewhat weaker xpeaks within aas (αn i,i ) plus long range NOES between strands, e.g. αα ij, αn ij, NN ij j i+1
Once beta-sheet is identified, can distinguish parallel from antiparallel àlook at long range NOES, i.e. between aas far apart in 1 sequence. Antiparallel beta-sheet: Cross strand NOEs αα ij ~2.3Å for alternate residues, strong NOE (long range NOEs) αn ij ~3.2Å NN ij ~3.3Å Parallel beta-sheet: Cross strand NOEs αα ij ~4.8Å v. weak (long range NOEs) αn ij ~3.0Å medium NN ij ~4.0Å weak Wuthrich, Billeter, Braum J. Mol. Biol. (1984) 180, 715-740
(2) Helix d NN (i,i+1) = 2.8Å for α-helix = 2.6Å for 3 10 -helix Primary feature of helix is a regular pattern of d NN (i,i+1) connectivities and d NN (i,i+2) ~4.1-4.2Å weak d αn (i,i+3) ~3.3-3.4Å
Summary of short distances in protein secondary structures From Wuthrich book, NMR of proteins and nucleic acids
Example: 45 aa synthetic peptide from UBA(2) of HHR23A Structure of a human DNA repair protein domain which interacts with HIV-1 Vpr, Dieckmann, et al, Nat. Struct. Biol. 5, 104 (1998) Also: observation of slowly exchanging amides corresponding to 2 structure H-bonds (not shown)