SAXS and collapse in protein folding Prof. Tobin Sosnick Univ. of Chicago with Jaby Jacob, Bryan Krantz, & Shane Dothager And P. Thiyagarajan (ANL) NIH and Packard Foundation for their generous support. Protein folding 1D Unique 3D Sequence Structure local structure long-range structure Vast conformational search over in milliseconds! HowHow toto reducereduce thethe ConformationalConformational Search?Search? Solve problem locally (e.g. Diffusion-collision model) collapse 2º Early Collapse (e.g. most simulations) collapse 2º Knowledge of when collapse occurs delineates folding models. Measure dimensions at early folding stages using SAXS BioCat beam-line, Advanced Photon Source, Argonne National Lab rapid mixing 2D detector apparatus (1-2 msec) θ APS ~20 µM focus 1 mm capillary 1E-5 ) Q I( 1E-6 1E-7 0.03 0.05 0.10 0.15 Q 2 2 −Q Rg 3 I(Q) = I 0e 1/width ∝ Rg Does the polypeptide collapse upon denaturant dilution? Rg ~ 26 Å 26 Å Dilute Denaturant Fold to completion or ~2 msec (takes 10+ msec) High denaturant <20 Å? Experimental strategy: Measure I(Q) for 0.4 s during continuous-flow period where protein has folded for ~2 msec. Ubiquitin in 1.5 M guanidine hydrochloride Sample concentration - 2 mg/ml Exposure time - 360 msec Number of exposures - 5 repeats for sample and buffer Guinier plots of refolding and refolded Ubiquitin Right after After folding has denaturant dilution completed (∼2 msec) (10+ msec) -4.0 -4.0 R =25.93 ± 0.03 Å g Rg=14.42 ± 0.01 Å Gdm = 0.75 Rg=26.5 ± 0.04 Å Gdm = 0.75 -4.5 Gdm = 1.5 -4.5 Rg=14.18 ± 0.01 Å Gdm = 1.5 ) -5.0 -1 ) -5.0 -1 m m (c c -5.5 Rg=26.6 ± 0.1 Å -5.5 ( Rg=16.67 ± 0.03 Å Gdm = 3.37 ) Gdm = 3.37 ) Q -6.0 )) ( -6.0 Q (I ( n -6.5 (I R =25.40 ± 0.07 Å L g n -6.5 Gdm = 4.5 L -7.0 R =27.6 ± 0.1 Å Rg=25.3 ± 0.2 Å g -7.0 Gdm = 6.0 Gdm = 4.69 Rg=25.9 ± 0.1 Å Gdm = 6.0 -7.5 -3 0.5 1.0 1.5 2.0 2.5 3.0 3.5x10 -7.5 -3 2 -2 2 4 6 8 10x10 Q (Å ) 2 -2 Q (Å ) Ubiquitin: Dilute denaturant Size after denaturant dilution Fold Size after equilibration Jacob et al, JMB, 2004) Ubiquitin (& ctAcp): Negligible collapse upon dilution of denaturant! Chemically denatured Proteins: Rg scales with length the same as a self-avoiding random walk 0.585 Rg = 2.1 N (Å) g R From K. Plaxco N Unfolded Ub & ctAcp fall on this line even under aqueous conditions (Ub: 26 versus 26.5 and ctAcp: 31 versus 30.7A) Polypeptides can behave like random coils under aqueous conditions No denaturant or Temperature dependence for Rg Equilibrium mode using non-folding polypeptide (RNase A with disulfide bonds broken) 50 50 40 40 30 30 (Å) g R 20 20 10 10 0 0 0123456 0 20406080100 [Gdm] Temperature (C) Increased strength of protein-protein Unfolded peptides are in the high interactions at lower denaturant temperature limit, where T∆S >> ∆H concentrations does not result in config compaction (protein-water (shrinking or expanding reduces number interactions are stronger). of conformations, i.e. entropy) Why is water such a good solvent for polypeptides? Hydrophobic interactions are strong (and will eventually drive folding to the native state for foldable sequences). But… 1. Collapse is inhibited by the loss of conformational entropy: 70 20 ΩU ~ 10 versus Ωcollapsed ~ 10 ∆G ~ 70 kcal mol-1 2. Removal of water from backbone is very demanding – Must satisfy H-bonding requirements. Each unsatisfied, buried amide and carbonyl costs several kcal mol-1 Net stability of a protein < 10 kcal mol-1 Therefore, non-specific hydrophobic collapse is unfavorable Conclusions (for small proteins which fold cooperatively) 1. Early collapse is not an obligatory step in folding 2. Water is a good solvent for unfolded chains (prior to the major folding event). 3. Polypeptides can behave like random coils in water Exceptions occur for larger proteins, and those with disulfide bonds or prosthetic groups. New area: RNA folding Ribozyme P RNA GA A 200 U A G A C G C U A A A Specificity 160 G A C U G P10.1 G U GG C G A C U AUA G A C U G U U G A G U U A G P12 U CU GC A S-domain G A C C A G G C C U U A C U G C C U U A G A 140 A G (~150 nt) A GG A U P11 A A A A G G C A A 220 G C A G AGGCUGA G C A C A C C A G U G GC U C P10 U U C U GA C G U 240 P5 P15 AA G CG A U P9 G A GCGAG AACC C U 120 G C U A A C GC U U 260 P8 U U P7GU C GCU C GGG U C A U A G G A G U A 60 U G A G G G G C U A C G G 280 C A A A 80C G A A P15.1 A U G C G A U C C CU U C U U U A A C G P5.1 C U C A G C U GGA A GA G U G C U U G AC 300 A 100 A G A A G A U GA G C A G A G A U A A U CU G A G U U C G A P18 G A 40 U G U P3 A A G GA G U AUCUGUA G 340 G A U G A 320 A G C U C U A GA C GUC G C G U G C G P4 C U 20 U A A U U G A U A G U A U P2 A U A C G G C 360 Catalytic 1 U G U P1 G C G C A P19 GUUCUU AA CGU U C GG C GC G U C-domain G AA A GA U U GC AA G A C A 400 C C A A (~255 nt) C U U C G G U A C A A U U C A 380 Model by Westhof & Co. JMB 1998 RNA Folding Rules are different Many highly collapsed intermediates C-domain 0 I (255 nucleotide 100 eq U 80kD, 0.3 mg/ml) 20 tion c 80 a ) p 40 m Å ( o 60 g 60 I nt c R Ik w/ Prof. Tao Pan N rce 80 1 Univ. of Chicago 40 Pe N 100 0.01 0.1 1 10 0 20406080From Fang et al, Mg2+ (mM) Time (s) Biochemistry, 2000 PNAS 2002 Uurea I k k N Ieq 1 I2 <10-3 s 30 s <1 s 5 s Relative compaction 0 94% 99% 100% Technical Issues: 1. Lower backgrounds i) reliably access lower Q ii) less sample iii) less potential aggreg’n iv) less sensitive to background subtraction 2. Detectors that can do time-resolved studies at high fluxes Often CCD’s take seconds to read-out. Hence, no “on-the-fly” studies on the subsecond time-scale (current experiments were conducted in continuous-flow mode) 3. Reproducibility – invariably need to take multiple acquisitions: Detector issues, beam movement? (lower backgrounds would help) “Many small, monomeric proteins fold with simple two-state kinetics and show wide variation in folding rates, from microseconds to seconds.” From S. Jackson Folding & Design 1998 How do small single-domain proteins fold? (see also Sosnick et al, NSB 1994, Sosnick et al, Proteins 1996, Krantz & Sosnick, Biochemistry 1999, Krantz et al. JMB 2002) Two-state folding ≡ only the U and N states populate All the energy and surface burial occur in the sole barrier crossing. Precludes formation of early collapsed intermediate. U N U I N Lack of an early collapse phase places constraints on computer simulations. short-lived, collapsed species t U fold Observ short-lived, post- transition state R ab intermediates g ) le (e.g. N time The black illustrative trace is consistent with ensemble behavior, as the observable probe has either the value of the unfolded state or native state for the majority of the trajectory. The blue and red trajectories are inconsistent with ensemble two-state folding behavior and the present SAXS data, as collapsed intermediates populate. Burst Rg and secondary structure formation (CD) Ub ctAcp 30 0 35 0 -1,000 ) -1,000 30 -1 25 -2,000 dmol -2,000 25 2 equil equil R -3,000 20 R g g burst (Å) 20 deg cm g burst -3,000 R R g R g -4,000 ] ( equil 2 15 equil 22 CD -4,000 15 CD burst -5,000 [θ CDburst CD 10 -5,000 10 -6,000 0123456 01234567 [GdmCl] (M) [Urea] (M) Burst phase CD signals: The removal of denaturant subtly alters the distribution of backbone dihedral φ,ψ angles, most likely resulting in a shift from the polyproline II region to the helical region of the Ramachandran map. Kratky and P(r) plots Qualitatively demonstrates absence of any significant collapse from the random coil conformation in the “burst phase” upon dilution to low denaturant 0.004 Equilibrium Burst phase 0.75M 0.75M 0.003 0.92M 1.50M 1.50M ) 3.75M 4.03M -1 Å 0.002 5.25M 4.69M ( 6.00M 6.00M r) P( 0.001 0.000 0 20406080100120 r (Å) Same result with CtACP: Negligible collapse upon dilution of denaturant Dilute denaturant Size after denaturant dilution Size after equilibration Folding times are msec to seconds – maybe we’re not looking fast No enough? Assume there is an early stable Energy conservation: ∆G ‡-∆G ‡=∆G u f intermediate Surface burial: mo=m +m ‡ observe u f ∆Gf fast ‡ observe ∆G ‡ ∆Gf u ‡ U I ∆Gu U ∆G N ∆G N m m mf u mf u o mo m ‡ ‡ Underestimate Energy : ∆Gu -∆Gf <∆G o And surface burial: m >mu+mf No missing energy, no missing surface burial - Therefore, the no early formation of a stable species which buries surface! Two-state Ubiquitin folding A) Chevron and amplitude data at 25 C, showing no rollover or missing amplitude.