a) Transportschden, sichtbar oder unsichtbar (Reklamationen fr solche Schden mssen umgehend bei der Transportfirma eingereicht werden)
Technische Daten A 4 Competition
Ausgangsleistung pro Kanal an 4 Ohm gemessen an 12 V. 4 x 60 Watt RMS Ausgangsleistung pro Kanal an 4 Ohm gemessen an 13,8 V. 4 x 85/150 Watt RMS/Musik Ausgangsleistung pro Kanal an 2 Ohm gemessen an 13,8 V. 4 x 153/250 Watt RMS/Musik Ausgangsleistung 1 Kanal gebrckt an 4 Ohm und 2 x 4 Ohm. 1 x 330/500 und 2 x 85/150 Watt RMS/Musik Ausgangsleistung 1 Kanal gebrckt an 2 Ohm und 2 x 2 Ohm. 1 x 480/800 und 2 x 125/200Watt RMS/Musik Frequenzbereich. 20 Hz - 20 kHz, +/- 0,3 dB Bassboost Pegelanhebung.0 - 9 dB Bassboost Einstellbereich der Mittenfrequenz. 30 - 120 Hz Frequenzweiche Regelbereich. 15 Hz - 7 kHz Pegelanhebung im 3-Kanalmodus der Kanle C und D.+/- 6 dB Klirrfaktor. < 0,009% TIM. < 0,016% Geruschspannungsabstand. > 100 dB Eingangsimpedanz. 10 kOhm Eingangsempfindlichkeit. 500 mV - 8 V Sicherung.3 x 25 Ampere Abmessungen (H x B x T) in mm. 35 x 240 x 432 Gewicht netto. 4,7 kg 5

Kanal B

Kanal B Channel C/D bridged auf on

Kanal C

Kanal D

4-Kanalbetrieb

Schalter 29
Kanal A und B gebrckt/ Mono
Kanal C und D gebrckt/ Mono
3-Kanalbetrieb Achtung! Im 3-Kanalbetrieb mssen die Kanle C und D gebrckt werden, da die Kanle A und B sich nur im 2-Kanalmodus brcken lassen. So ist gewhrleistet das die Kanle C und D als Monokanal fr den Subwooferbetrieb benutzt werden, da diesem auch die Bassanhebung zugeordnet ist.

Schalter 30

2-Kanalbetrieb Achtung! Fr den 2-Kanalbetrieb mssen zuerst die Kanle C un D gebrckt werden. Sollten sich die Kanle A und B nicht brcken lassen, berprfen Sie, ob der Schalter 29 der Kanle C und D auf bridged on steht.
Vollaktiver Betriebsmodus Mit Hilfe des Schalters 30 kann der Verstrker A4 zum Aufbau eines vollaktiven Systems genutzt werden. Hierzu wird der Schalter 30 Full Active Operating Modeauf Position on gestellt. Es wird immer nur das Tiefpass-Signal zu den Kanlen C und D weitergeleitet.
Dear Customer, congratulations on your purchase of this high-quality HELIX amplifier, made in Germany. The new Helix competition series amplifiers highlights best quality, excellent manufacturing and state-of-theart technology. After 23 years of experiences in the research & development of audio products this amplifier generation sets new standards. The attractive typical Helix design makes this amplifier to an outstanding and top of the class product. We wish you many hours of enjoyment with your new HELIX amplifier. Yours AUDIOTEC FISCHER Team Before drilling the holes for the screws, carefully examine the area around the installation position and make sure that there are no electrical cables or components, hydraulic brake lines or any part of the petrol tank located behind the mounting surface - otherwise these could be damaged. You should be aware of the fact that such components may also be concealed in the double-skin trim panels/mouldings. General instruction for connecting the amplifiers The HELIX amplifiers may only be installed in motor vehicles which have a 12-volt minus pole connected to the chassis ground. Any other system could cause damage to the amplifier and the electrical system of the vehicle.

General installation instructions for HELIX amplifiers To find out how HELIX amplifiers work best for you, read this manual carefully and follow the instructions for installation. We guarantee that this product has been checked for proper functioning before shipping. Before you start installation, disconnect the car battery at the minus pole. We would urge you to have the installation work carried out by a specialist as verification of correct installation and connection of the unit is a prerequisite for warranty cover of the HELIX amplifier. Install your amplifier at a dry location where there is sufficient air circulation to ensure adequate cooling of the equipment. For safety reasons, the amplifier must be secured in a professional manner. This is performed by means of four fixing screws screwed into a mounting surface offering sufficient retention and stability. 6
The plus cable from the battery for the complete system should be provided with a main fuse at a distance of max. 30 cm from the battery. The value of the fuse is calculated from the maximum total current input of the car audio system. Install the cabling in a manner which precludes any danger of the leads being exposed to shear, crushing or rupture forces. If there are sharp edges in the vicinity (e.g. holes in the bodywork) all cables must be cushioned and protected to prevent fraying.
Never lay the power supply cables adjacent to leads and lines connecting other vehicle equipment (fan motors, fire detection modules, gas lines etc.). In order to ensure safe installation, use only highquality connections and materials. Ask your dealer for high quality accessories.
4 Fuses and D 7-10 Signal input nel A and B nel C and D
1 Connecting the remote lead 2 Connecting the battery cable 3 Connecting the ground cable 5 Fuse Function Indication 6 Input Signal Switch for Inputs C
20-21 Control for frequency range 22-23 Control for level adjustment 24 Speaker terminal for channel A and B 25 Speaker terminal for channel C and D 26 Control to raise the center frequency 27 Adjustment of center frequencies 28-29 Mono/ stereo switch 30 Full active operating mode 31-32 Level control for channel C and D in full active operating mode
11-14 Level controls for input sensitivity 15-17 CPS-Colour Protection System 18 Switch for HP/ Linear/ LP of chan19 Switch for HP/ Linear/ LP of chan-

ENGLISH

Position 1: Lowpass

Position 3: Lowpass

variable
1 Connecting the remote lead The remote lead is connected to the automatic antenna (aerial positive) output of the head unit (radio). This is only activated if the head unit is switched ON. Thus the amplifier is switched on and off with the head unit. 2 Connecting the battery cable Connect the +12 V power cable to the positive terminal of the battery. Recommended cross section: min. 16mm_. 3 Connecting the ground cable The ground cable should be connected to a central ground reference point (this is located where the negative terminal of the battery is grounded at the metal body of the vehicle), or to a bright bare-metal location on the vehicle chassis, i.e. an area which has been cleaned of all paint residues. 4 Fuses The input fuses are connected in parallel and provide protection against an internal equipment fault, i.e. the system must be additionally protected by a further line fuse located in the vicinity of the battery (max. distance from battery: 30 cm). The fuse ratings are 3 x 25 amperes, and both must be installed as the amplifier protection rating is 75 amperes (3 x 25 A). 5 Fuse Function Indication If the fuses (4) are destroyed due to malfunction the red LED illuminates. In normal operation the LED turns off. 6 Input Signal Switch for Inputs C and D If there is only a mono signal in bridged mode on channel C and D the inputs can be connected with switch (6) on position 2. A Y-adapter is not necessary. Trimode is only possible when channels C and D are bridged. Drive channel C and D individual on position 1.

7-10 Signal input The A4 Competition has RCA connectors for RCA cables that can be connected with the pre-amplifier output of the line-outputs of the headunit or of a pre-amplifier i.g. HXE 100. This connectors are gold-plated to ensure a better signal transmission. 11 - 14 Level controls for input sensitivity These controls can be used to match the input sensitivity of the individual channels - A to D - to the output voltage of the connected head unit. These controls are not volume controls and are solely intended for the purpose of sensitivity trimming. The control range extends from 700 mV to 8 V.

Position 1: Highpass

15-17 CPS - Color Protection System This LEDs show the operation mode of the amplifier green = in operation; yellow = malfunction of the amplifier, short circuit at loudspeaker output; red = overheating If the amplifier shuts off due to overheating it can take a while until it turns on again. This depends on the outside temperature. 18 Switch for HP/ Linear/ LP of channel A and B To switch over the internal active crossover to highpass/ full range (linear) or lowpass of channel A and B 19 Switch for HP/ Linear/ LP of channel C and D To switch over the internal active crossover to highpass/ full range (linear) or lowpass of channel C and D

Position 2: Lowpass

Hz Hz 800 Hz variable

Position 3: Highpass

22 - 23 Control for level adjustment Control to adjust the crossover frequency of stereo channel A and B (22) and C and D (23) 24 Speaker terminal for channel A and B 25 Speaker terminal for channel C and D 26 Control to raise the center frequency This control enables the center frequency set at control 27 to be raised from 0 to 9 dB if the switch 19 is on HP or LP position.

Position 2: Highpass

20 - 21 Control for frequency range This control enables to adjust the control range of the potentiometer 22 (channel A and B) and 23 (channel C and D) from 15 Hz up to 90 Hz on position 1, from 90 Hz up to 800 Hz on position 2 and from 600 Hz up to 7 kHz on position 3.
27 Adjustment of center frequencies Control 27 can be used to select the frequency from 30120 Hz of the adjusted lowpass (control 23). this can be enhanced with control 26 from 0 to 9 dB. It is usefull to emphasize or correct a determined frequency of the subwoofer (kickbass)
Caution: Bridge at first channel C and D before you switch to 2-channel mode. If channel A and B cannot be bridged make sure that control 29 of channel C and D is set on position 2 (mono). Only input A and B have to be used in 2-channel mode. Thus the signal of input A is used for the bridged channel A and B and the signal of input B for the bridged channel C and D. 30 Full active operating mode With the use of control 30 the amplifier is operating in full active mode. Set the switch on position 2. In this case the frequencies of input A and B (only this can be used in full active mode) are set for channel A and B. The lowpass of the signal's adjusted frequency with control 22 for channel A and B will be transmitted to channel C and D, regardless of switch 18 position (HP/ Lin/ TP), no matter if channel A and B are used as highpass, full range or lowpass, the signal is always transmitted as lowpass to channel C and D. Adjust the desired frequency of the transmitted signal with control 22. Due to the transmission of lowpass to channel C and D it is possible to build a bandpass with a bandwidth of 15 Hz up to 7 kHz.

variable Hz 500

120 Hz
28 - 29 Mono/ stereo switch To set the operating mode of the amplifier. 4-channel: If the amplifier operates in 4-channel mode both switches have to be set on stereo and position 1 3-channel: If the amplifier is operates in3-channel mode (frontsystem/ subwoofer) set control 28 of channel A and B for the frontsystem on stereo, position 1 and control 29 of channel C and D on mono, position 2. Caution: Channel C and D must be bridged in 3channel mode operation, because channel A and B can only be bridged in 2-channel mode. It ensures that channel C and D are used as mono channel for the subwoofer. Only this channels have bassboost. Both inputs 9 and 10 must be used for bridged channel because both channels operate the summation signal. If there is only one mono signal for the subwoofer the signal must be distributed on both inputs with switch 6 or use a Y-adapter. 2-channel: If the amplifier operates in 2-channel mode set control 28 of channel A and B and switch 29 of channel C and D on mono, position 2.
Example 1: When channel A and B are operating in full range channel C and D have the lowpass frequencies set with control 22. Example 2: When channel A and B are running as highpass 2 kHz, the frequencies up to 2 kHz are for channel C and D. If you turn to highpass i.e. 80 Hz you get a bandpass fro 80 Hz to 2 kHz for channel C and D.

variable 2K

31- 32 Level control for channel C and D in full active operating mode Adapt the output level of channel C and D with control 31 (channel C) and 32 (channel D) in full active operating mode to channel A and B. The control range of each channel is +/- 6 dB. In 3-channel mode (mono C/D) both controls (31/32) have to be used. In 2 channel mode these controls are not activated.
Caution! To avoid a lost of sound pressure make sure that the crossover frequencies of high-and lowpass are separated of 2 Octave when building a bandpass on channels C/D in full active mode. That means: If the lowpass signal of channel A/B is transmitted to channel C/D, the highpass of channel C/D should be 2 Octave lower. Example: Lowpass tansmission 320 Hz - then highpass crossover frequency adjustment on channel C/D = 80 Hz (1 Octave = double frequency or halve frequency).
Never connect the loudspeaker cables with the car chassis gound. It damages your amplifier. Ensure that the loudspeaker systems are correctly connected (phase), i.e. plus to plus and minus to minus. The plus pole is indicated on most speakers. The A 4 is adjusted as 4-channel amplifier before shipping. In tri-mode operation channel C and D must be bridged to mono. In most of the installations the bridged mono channel due to the higher output power is used to run the subwoofer and the bassboost is assigned to this channels. In 2-channel operating mode channel A and B can only be bridged when C and D are already bridged.

Bandpass 80Hz to 2 kHz

Important Notice: The amplifier A4 Competition has a temperature controlled innovative power supply. This newly developed feature makes possible that the amplifier reduces the output power at very high temperature. It protects the amplifier from destruction and overheating shut off. This power reduction is not audible in normal application because it only comes into affect at high power peaks and when the amplifier gets very hot due to high outside temperatures. If the amplifier shuts off due to overheating (> 95C) it can take some minutes until it turns on again. Also due to the integrated safety electronic the amplifier turns on with a delay of 3-4 seconds. This is because of the internal check up of all relevant voltages that ensure a perfect operation. This amplifier has several electronic protection circuits that shut off the amplifier at overheating, overloading, short-circuit on loudspeaker, low-ohmic mode or defective power supply. It is indicated through different LEDs (see CPS system at point 15-17). Please check for connecting failures such as short-circuits, wrong connections and over-temperature. If the amplifier does not turn on it is defect and has to be send to your local authorized dealer for repair service. A detailed description of the malfunction and the purchase receipt has to be attached.
Important informations for connecting loudspeakers Die Helix A4 Competition has 4 integrated power amplifiers. Each of them is 1 ohm stable. That means a loudspeaker impedance can be connected on each channel. In bridged mode two each amplifiers are working together as one channel. In this case pay attention that each channel has to process half of the connected impedance. Example: If a 4 ohms loudspeaker is connected to a bridged amplifier, it means 2 ohms for each channel and so 1 ohm with a 2 ohms loudspeaker. Therefore 2 ohms is the lowest impedance that can be connected to a bridged pair of channels, because each single channel is 1 ohm stable. Do not run all channels with an impedance of 1 ohm because the total current is getting too high. In this case it can be that the calculated total power of the amplifier is going beyond and the input fuse or the amplifier will be destroyed. It makes no sence to connect one ohm on all channels because the total loss of power will be too high, that means the power will not be transmitted by the loudspeaker, it will convert into heat within the amplifier. To ensure proper function of the A4 Competition amplifier please pay attention on the following rules: 1. The total impedance of all connected loudspeakers on all channels should not be lower than 6 ohms, no matter if a pair of channel is bridged or not. Some examples: 4 x 1.5 ohms or 1 x 2 ohms (bridged) + 2 x 2 ohms or 2 x 1 ohm + 1 x 4 ohms (bridged) or 2 x 3 ohms (bridged) etc. The total impedance of all examples is not lower than 6 ohms. If two 2 ohms woofer are connected (total 4 ohms) on two bridged channels or ohm loudspeaker (total 4 ohms) are connected on each channel and the total impedance is below 6 ohms the amplifier can shut off, the fuses can burn or the amplifier can be destroyed. 2. The impedance per channel should not be lower than 1 ohm.

We guarantee a lot of pleasure with the A4 Competition if you take notice of this informations.
date this warranty. 4) Consult your authorized dealerr first, if warranty service is needed. Should it be necessary to return the product to the factory, please insure that a) the product is packed in original factory packing in good condition b) the warranty card has been filled out and attached to the product c) the product is shipped prepaid, i.e. at your expense and risk d) the receipt/invoice as proof of purchase is enclosed 5) Excluded from the warranty are: a) Shipping damages, either readily apparent or concea-

Warranty Regulation

Due to the high quality standard Helix products achieved an excellent international reputation. Therefore we grant a warranty period of 2 years.
The products checked and tested carefully during the entire production process. In the case of service note the following: 1) The 2 years warranty period commences with the purchase of the product and is applicable only to the original owner.
2) During the warranty period we will rectify any defects due to faulty material or workmanship by replacing or repairing the defective part at our decission. Further claims, and in particular those for price reduction, cancellation of sale, compensation for damages or subsequential damages, are excluded. The warranty period is not altered by the fact that we have carried out warranty work. 3) Unauthorized tampering with the product will invali-
Technical Data A 4 Competition
Cont. power rating at 4 Ohms per channel measured at 12 V. 4 x 60 Watt RMS
Cont. power rating at 4 Ohms per channel measured at1 13,8 V. 4 x 85/150 Watt RMS/Musik Cont. power rating at 4 Ohms per channel measured at1 13,8 V. 4 x 153/250 Watt RMS/Musik Cont. power rating at 1 ch. bridged at 4 Ohms and 2 x 4 Ohms. 1 x 330/500 und 2 x 85/150 Watt RMS/Musik Cont. power rating at 1 ch. bridged at 2 Ohms and 2 x 2 Ohm. 1 x 480/800 und 2 x 125/200Watt RMS/Musik Frequency response. 20 Hz - 20 kHz, +/- 0,3 dB Bassboost.0 - 9 dB Bassboost setting range for center ferquencies. 30 - 120 Hz active crossover frequencies. 15 Hz - 7 kHz Level sensitivity in 3-channel mode for the channels C and D.+/- 6 dB Total harmonic distortion (THD. < 0,009% TIM distortion. < 0,016% Signal to noise ratio. > 100 dB Input impedance. 10 kOhm Input sensitivity. 500 mV - 8 V Fuse.3 x 25 Ampere Dimensions (H x W x D) in mm. 35 x 240 x 432 Weight net. 4,7 kg 9

led (claims for such damages must be immediately notified to the forwarding agent). Scratches in metal parts, front panels or covers etc. This must be notified to your dealer within 5 days of purchase. Defects caused by incorrect installation or connection, by operation errors, by overloading or by external force. Products which have been repaired incorrectly or modified or where the product has been opened by other persons than us. Consoquential damages to other equipments. Reimbursement when repairing damages by third parties without our previous permission.

Channel A

Channel B
Channel B Channel C/D bridged on on

Channel C

Channel D

4-channel

switch 29
Channel A and B bridged/ Mono
3-channel Caution: Channel C and D must be bridged in 3-channel mode operation, because channel A and B can only be bridged in 2-channel mode. It ensures that channel C and D are used as mono channel for the subwoofer. Only this channels have bassboost.

switch 30

Channel C and D bridged/ Mono
2-channel Caution: Bridge at first channel C and D before you switch to 2-channel mode. If channel A and B cannot be bridged make sure that switch 29 of channel C and D is set on position bridged on.
Full active operating mode With the use of control 30 the amplifier is operating in full active mode.Set the switch 30 Full Active Operating Mode to position on. The signal is always transmitted as lowpass to channel C and D.
AUDIOTEC FISCHER GMBH Gewerbegebiet Lake II Hnegrben 26 D-57392 Schmallenberg Tel.: ++49 (0) 29 72-0 Fax: ++49 (0) 29 72-88 E-mail: info@audiotec-fischer.com Internet: www.audiotec-fischer.com

ARTICLE IN PRESS

doi:10.1016/j.jmb.2006.02.026 J. Mol. Biol. (2006) xx, 18

C OMMUNICATION

Alternate Pathways for Folding in the Flavodoxin Fold Family Revealed by a Nucleation-growth Model
Erik D. Nelson* and Nick V. Grishin
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center 6001 Forest Park Blvd., Room ND10.124, Dallas, TX 75235-9050, USA A recent study of experimental results for avodoxin-like folds suggests that proteins from this family may exhibit a similar, signature pattern of folding intermediates. We study the folding landscapes of three proteins from the avodoxin family (CheY, apoavodoxin, and cutinase) using a simple nucleation and growth model that accurately describes both experimental and simulation results for the transition state structure, and the structure of on-pathway and misfolded intermediates for CheY. Although the landscape features of these proteins agree in basic ways with the results of the study, the simulations exhibit a range of folding behaviours consistent with two alternate folding routes corresponding to nucleation and growth from either side of the central b-strand.
q 2006 Published by Elsevier Ltd. *Corresponding author
Keywords: fold families; equilibrium intermediates; non-native interactions
From a folding perspective, the topology of a protein is interpreted by the shape of its native backbone which loosely determines the pattern of atom-to-atom cross-links between its amino acid residues. Over the past several years, simple theoretical and computational models based essentially on topology and minimal entropy loss13 have demonstrated that native topology is a rst order effect deciding the way a protein folds.412 While the data so far still provide a very incomplete picture, it suggests that if we could provide any consistent description of protein folding it would be that evolutionary changes which, roughly speaking, conserve topology1315 and act as perturbations affecting mainly the depths of intermediates and the heights of free energy barriers on a proteins folding landscape rather than the basic mechanism1618 that allows it to fold. However, among these results have now appeared a growing number of excursions away from axiomatic correspondence between folding and topology that must somehow nd a place within this picture.1924 For example, the small proteins L and G share an almost identical, symmetric topology, but both proteins nucleate one of their two b-sheets preferentially, breaking the symmetry of the native fold.20,21 The small, all-helical proteins Im7 and Im9 share essentially
E-mail address of the corresponding author: enelson@spirit.sdsc.edu
0022-2836/$ - see front matter q 2006 Published by Elsevier Ltd.
the same topology, but Im7 folds through an onpathway intermediate in which a distorted arrangement of its helices is stabilised by non-native interactions.22,23 Perhaps, it is not so surprising that the folding mechanisms of these proteins are varied. Their native shapes are not frustrated mechanically7 so they should have greater freedom to respond to structural and energetic perturbations, and their responses (the modulation of intermediates and pathways by these perturbations) may even be somewhat continuous. On the other hand, even small perturbations such as amino acid substitutions can sometimes cause discrete interconversions of protein structure within a fold family (for instance, changing b-strands to b-helices24,25). Moreover, the structural family of a protein (its fold type or fold classication) often allows large loop insertions, sometimes within secondary structure units, and the substitution of one secondary structure type for another, all of which can affect the entropy of its folding units, the pattern of native contacts between them, and the capacity of these units to evolve more favourable contacts. Accordingly, this more exible interpretation of topology (fold type) should permit more substantial variations to occur among protein folding mechanisms. The landscape features that dene the folding pathways of larger proteins (w200 amino acid residues) are more discrete, and should have more capacity to accommodate perturbations. These

2 features still appear to be guided by native topology,5,26 however, given the larger and less predictable variations in structure that can be admitted into the fold families of larger proteins, a manifestly pathway-like protein could conceal, in an evolutionary sense, alternate folding routes due to multiple folding units that are responsive to preferential stabilization by a suitable accumulation of these perturbations. Therefore, as with proteins L and G, a purely structural classication of protein families can permit substantial variations among the folding routes of a given fold type, but for larger proteins this may start to dene discrete spectra of mechanical differences, or modes for folding within a family. If multiple routes do exist for a particular fold type, when does nature choose from among them, and when does it admit mixtures of the routes? These types of problems are just now beginning to be explored,19,27 and they are of interest not simply in terms of the physics of how proteins fold but because they may provide information about low lying conformational sub-states that decide how proteins function. Because of the complexities involved in obtaining this information experimentally, simple, computationally efcient folding models, such as those recently used to describe protein transition state structures2837 could be very useful to infer folding properties and thus direct the process of these measurements more effectively. Here, we use one of these models for a detailed exploration of CheY and two other large proteins from the avodoxin fold family.27 The model is one of an extremely simple type in which amino acid residues are allowed to exist in just two states, either folded (frozen) or unfolded (a discussion of the model is given in the Appendix). Its energetics are heterogeneous and Go-like, the interaction between any two amino acid residues being proportional to the number of atom-to-atom contacts that would exist between them in the native crystal structure of the protein. Each collective state of the amino acid residues is intended to represent a small micro-ensemble consisting of the conformational states of unfolded segments constrained by the frozen amino acid residues and the cross-links that form between them. The entropy of the micro-ensembles is described using simple estimates from polymer theory in which the unfolded segments are modelled as random ight (gaussian) chains and only the space occupied by frozen parts of the molecule is excluded. In current applications of this model,3134 the micro-ensembles are limited to very simple objects (for example, a nucleus or nuclei with two or fewer loops) for the sake of simplifying the computations. However, it is known that these approximations ~ begin to break down around O100 amino acid residues, precisely where the ne scale features of folding start to matter less and where, due to its speed, the model could be of most use. In a recent paper,36 we developed an approach to sample more

Alternate Pathways for Flavodoxin Folding
complex micro-ensemble topologies excluded in previous work in order to investigate larger proteins with multiple folding units. We found that including these topologies often led to qualitative improvements in the calculation of transition state structure, and that the dominantly occurring micro-ensembles turned out to have a simple scaling form (see the Appendix) for which an explicit calculation of excluded volume effects38 of the type noted above would not be too forbidding. Although we account for these effects in only an order of magnitude sort of way, this approximation seems to be enough to draw the kinds of conclusions we need for this work. The CheY topology studied here seems particularly well suited to description by this model. The transition state structure of CheY (3chy.pdb) compares relatively well with available protein engineering data39,40,43 (correlation coefcient 0.62 or 0.94 if volume increasing mutations are excluded) and the model detects the misfolded and on-pathway intermediate states thought to reect topological frustration7,27 between interior (b-sheet) and exterior (a-helix) layers of the fold that bridge two weakly interpenetrating domains43 on either side of the central b3 strand. The level of agreement is surprising since the misfolded intermediate4,39,40 is thought to result from the dynamical connection between these layers and lead to a non-native distortion of the helices, yet we observe the intermediate in a model without explicit dynamical constraints and native-only interactions (Figures 1 and 2). On-pathway the agreement is surprising as well. In crossing the transition state, CheY nucleates from its N-terminal domain and growth is thought to proceed by strands of the b-sheet which frustrates the accretion of a-helices onto the exterior. Again, this is exactly what we observe in our simulations. In rough agreement with the experimental results of Lopez-Hernandez & Serrano,40,43 the nuclear region includes b1-a1-b2 and part of a2 (we refer to regions on either side of the central b3 strand as domains A and B below). The minima in the free energy prole (Figure 2) register with the formation of b-strands and the helices start to form just before the maxima so that the conict in stability between interior and exterior regions of the fold is periodically resolved in crossing the barriers. The unusual unfolding and refolding features of helices a4 and a5 in Figure 1(a) and the accentuation of the intermediate barrier after b4 in Figure 2 may signify non-native interactions in the actual folding path as we explain later below. The avodoxin study of Bollen & van Mierlo27 suggests that proteins from the same fold family (CheY, cutinase and anabaena apoavodoxin in this instance) may exhibit a similar pattern of on and offpathway intermediates. These proteins have lengths ranging from 128 to 197 amino acid residues and very low sequence identity, and protein engineering results exist only for the smallest member, CheY. Interestingly, both cutinase

Figure 1. Projection of the folding landscape onto (a) a-helices and (b) b-strands for CheY. Pn(q) is the probablity that sub-structure n (helix or strand 15) is folded when there are q frozen amino acid residues. The folding process is stepwise, nucleated by domain A (b1-a1-b2) at qw38 and proceeding to accrete each section, anbnC1, in order along the interior b-sheet. After the protein is nucleated, the addition of each new helix (strand) leads to a maxima (minima) in the free energy prole (Figure 2). The misfolded helical intermediate observed by Clemente and co-workers is detected near qw24. Across this region, the strands and loops in domain B remain unfolded totally, the number of nuclei jumps (the probability of four nuclei reaching about 0.1 at qZ24) and the distribution of nuclear sizes changes abruptly from bimodal (distributed about 2 and q amino acid residues) to unimodal (distributed about two amino acid residues, the segment size used in the simulations) to bimodal before reaching the transition state. (c) Ribbon diagram of the CheY crystal structure. Light blue regions indicate amino acid residues with native contacts dened by Nelson & Grishin7 and Shea et al.10
(1agy.pdb) and apoavodoxin (1ftg.pdb) contain a number of exible loop insertions (in cutinase these include a-helical fragments) at points where a-helices would connect to b-strands in the B (Cterminal) domain of CheY. These insertions could relax the interiorexterior frustration effect suggested by these authors and allow for greater stability of the B domain which could lead to variations among avodoxin fold pathways. Our results for cutinase and apoavodoxin do share many of the same features described for CheY. Like CheY, the key folding event is growth of the nucleus up to and across the b3 strand dividing the A and B domains of the fold. Also, each protein exhibits, to varying degrees, the signal of a misfolded intermediate in which helices but not strands or loops (except in the nucleus) are folded, and minima (maxima) in the landscape register with the formation of b-strands (a-helices) consistent with frustration between the interior and exterior regions of the protein. However, at least for apoavodoxin, the structural mechanism for folding is quite different. The nucleus of apoavodoxin is on the opposite side

the segments unfold near qZL/2 (L is the length of the protein) and are simultaneously replaced by domain A, a2, and b3 before the reaction proceeds. The folding plots have an all or none character that suggests the exchange of B-like for A-like nuclei is part of the folding pathway27 even though the molecule begins this process from a partially misfolded state. Aside from structural processes, the results above appear roughly consistent with the experimental data. The sizes of free energy barriers are comparable in scale to the results reported by Bollen & van Mierlo, and although it is difcult to establish the topography of the landscape near the misfolded intermediate, the proles seem as if they could be classied in a similar way. For example, the CheY kinetics were analysed with both on and offpathway models by the Serrano group to indicate that they lead to the same results.40 This is consistent with the fact that the main transition state can be reached by a partial exchange of helical structure in domain B for nuclear structure in domain A as is indicated by our own results. However, in apoavodoxin and cutinase domain B folds rst, so the exchange should be qualitatively different, and this may explain why an off-pathway kinetic model27 could describe these two experiments better. Does this over-simplied model predict the basic signature of the folding landscapes? The model appears to be operating as intended. (i) The transition state structure of the CheY topology agrees well with experiment. (ii) Complex diagrams (nested, inter-linked loop, etc.36) are very infrequent in simulations for this fold type. (iii) There are very few contacts between domain A and domain B (after strand b3) so the nuclei in these two regions are free to fold in parallel (see the Appendix).
Figure 3. Projection of the folding landscape onto (a) a-helices and (b) b-strands for apoavodoxin. The helix indices follow the crystal structure data in which a2-a3 corresponds to the CheY helix a2. The strand indices are the same in all three of the avodoxin proteins. The nucleus of apoavodoxin includes part of the C-terminal helix a6, all of b5, most of a large loop l6 preceeding, or inserted into b5, a small part of helix a5 and the loop preceeding it (see Figure 4). As the transition state is crossed, the rest of a6 forms, and folding continues to alternate from a to b moving from C to N-terminal ends until the protein is folded. Again, there are two minima (basins) in folded wing of the free energy prole, comparable in size to CheY, that register with the formation of bn-anK1 layers. The signature of an intermediate with helical structure is visible near qZ36.

Figure 4. Prole of atom-to-atom contacts, ci, for (a) CheY and (b) apoavodoxin. ci is the number of atom-to-atom contacts with amino acid i divided by the mean (a contact is registered when atoms from non-nearest neighbor amino acids are less than 5 A apart; the Figure is coarse grained in blocks of two amino acids). Shaded bars in the lower part of the Figures indicate the nuclear regions. For each fold, the number of contacts between domain A and the nuclear part of domain B (after the dividing b3 strand) is about the same as that for two amino acids. The local accumulations of contacts and the opposing slopes of the proles coincide with the location of nuclei and the direction of their growth.
(iv) The patterns of atom-to-atom contacts are consistent with the way each protein folds, and although the entropy cost to freeze unfolded segments of proteins depends on amino acid composition,25 it seems unlikely that including this dependence could lead to something concerted enough to reverse the effect in Figure 4. Finally, (v) in mechanical unfolding7 of apoavodoxin, both domain A (the CheY nuclear region) and the helixstrand combination a6-b5 in domain B (the apoavodoxin nucleus) are dynamically conned by their local environments, moving essentially as xed units while the protein unfolds and remaining so long after the core of the protein is exposed to solvent. As we noted above, non-native interactions can have a substantial impact on, or even control the folding of certain proteins, and some of our results seem to suggest these effects. Although the model does not include non-native interactions directly, proteins do, and the results may reect their absence in the model at certain points along the folding proles. The effects of non-native interactions have never been looked at using this type of model and hence it is difcult to decide when they could be present, or what signature they would leave on the model kinetics. Consequently, we decided to look at the Im7 folding landscape where these effects have been mapped out.22,23 Im7 folds through a single intermediate in which three of its four helices (a1, a2, and a4) are structured but distorted non-natively, maximizing the burial of hydrophobic side-chains that would be exposed had the helices adopted their native positions. In crossing the transition state into the native fold, the helices acquire their native orientations, and the binding site for helix a3 is exposed allowing it to fold and ultimately lock the whole protein into its native structure. Our results for Im7 are shown in Figure 5. Its sister protein, Im9, folds across

a smooth free energy barrier but still shows some indications of an intermediate perhaps suggesting the results seen at low pH.23 Both proteins condense into relatively large, partially unfolded ensembles (Figure 5(b)) due to exposed side-chains in the turn regions of the folds. This situation can be improved a bit by extending the contact radii or by including the dependence of the entropy on amino acid type, however, the results here are still very instructive. Again, in the intermediate parts of the protein are stabilized by non-native interactions. When the transition state is crossed, these stabilizing contacts are exchanged for native contacts and the energetics of the protein and the model converge. Any qualitative differences that exist between the model and the protein due to the missing nonnative interactions should be evident before the transition region where these interactions are lost and the differences between the two pathways are reversed. Regions of the protein that are stabilized by non-native interactions in the intermediate should be less stable in the model and may tend to fold late, while regions that are not stabilized by these interactions would tend to fold early. Because this behaviour is reversed on crossing the transition state, it should be evident (if the effect is strong enough) as some type of wrinkle in the time order for folding the sub-structures involved in the intermediate, and this is exactly what we observe. The folding order for sub-structures in the protein and the model converge on the right side of the transition barrier just after the major intermediate (we refer to this point as q* in Figure 5(b)). Within the model intermediate, helices a1 and a2 are structured, and as the transition barrier is crossed helix a3 folds, unfolds, and then refolds after helix a4 converging with the experiments. The barrier is a residue of the competition between (i) the free energy of freezing helix a4 leaving the loop including helix a3 unfolded and

(a) 1 4

(b) q*
Figure 5. Projection of the folding landscape onto (a) helices and (b) the free energy prole for Im7. The helix probabilities, Pn(q), qualitatively agree with the experimental results in the region qRq* where helix 3 starts to fold onto the nucleus consisting of helices 1, 2 and 4. In the intermediate preceeding this region, helices 1 and 2 are folded. As the barrier is crossed, helix 3 initially folds onto helices 1 and 2, then unfolds and is replaced by helix 4, and nally refolds to complete the reaction.

(ii) the free energy of freezing a3 with a4 unfolded. Apparently, forming the loop by native interactions only is unfavourable in both the protein and the model, but the protein can avoid this situation by escaping into the non-native dimension of the free energy landscape41 where it nds more favourable contacts. The model initially folds helix a3 rst, but when the model and protein pathways begin to converge near q*, the stabilizing energy of the protein transiently compensates the a3 loop. The effects of non-native interactions therefore emerge here as a consequence of the lower dimensionality of the model.41 Whatever conformations are dynamically accessible to a protein and are somehow stabilized that are not available to the model protein will be subject to the effects described above. To some degree, it should be possible to infer the existence of non-native intermediates from the time reversal of domain stabilities, however, even for Im7 it is difcult to decode the actual sequence of folding events from Pn(q) alone. Thus, although the exchange region in cutinase resembles the transition in Im7, the results could be explained equally well by some inherent frustration in the protein. On the other hand, the CheY on-pathway intermediate involves sub-structures that surround a small pocket, or cavity in the fold, and it seems possible that helix a5 could initially pack nonnatively onto the protein with helix a4 partially unfolded (very similar to a4 and a3 in Im7) to better stabilize this part of the fold. In summary, although these uctuations, or timeorder wrinkles do not provide an absolute test for non-native interactions, their absence suggests that native topology is the dominant effect that guides the folding process. Consequently, for all of the reasons cited above, we believe our results demonstrate that, at the very least, avodoxin-like folds permit alternate folding pathways corresponding to nucleation from either side of the central b3 strand. If this result turns out to be true, an interesting
subject for experiment would be to nd out whether these pathways can exist in parallel (so that both ends fold and join in the middle) or whether, as it now appears, there is some structural reason why one end or the other of avodoxin-like topologies tends to fold preferentially. Looking back on our results, it is remarkable that this system, which is essentially just an Ising model with non-local interactions, can distinguish among the kinetic attributes of these extremely complex objects. The fact that complicated features such as the off-pathway intermediate in CheY and the dynamical connement of nuclear regions in apoavodoxin can be detected by a theory that essentially consists of contact weighted native cross-links and loop closure entropy indicates a very basic connection between the structural attributes of local regions in a protein (their shape and connectivity to the rest of the protein) and how such regions are organized dynamically, which ultimately decides the different ways proteins can fold.

Supplementary Data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. jmb.2006.02.026

Appendix

The justication of using this type of model to study proteins as something even minimally reasonable extends from the work of Munoz & Eaton where it was directly applied to quantitatively describe the folding of a small b-hairpin molecule and later predict the folding rates of small two-state proteins.2931 The approach we describe here is an extension of a separate approach33,34 that
7 proteins studied so far (staphylococcal nuclease) and plays at most a very limited part in the systems investigated here.
provided a starting point to account for the excluded volume effects decribed in the text. The free energy of a micro-ensemble g in Galzitskaya & Finkelstein33 is dened as: Fg Z e

g X i!j

" di; jKT LKqs C

# sp (A1)

References
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where in the rst (energetic) part of this expression, d(i,j) is the number of heavy atom contacts (including main-chain atoms) between residues i and j in the native crystal structure and the sum P i!j g includes all pairs of amino acids that are frozen in g. In the entropic part of the expression, L is the chain length, q is the number of folded residues, sZ2.3R is the entropy cost to freeze an amino acid, and s(p) is the entropy cost to link the ends of an unfolded segment into a loop p. Unfolded ends and open segments are described as free chain segments while s(p) is approximated by a gaussian chain with ends attached to an impenetrable surface (in the work done by Galzitskaya & Finkelstein,33 the number of loops and or open segments in a micro-state is limited to two). The energy scale e is set by performing the experiments at equilibrium between native (qZ1) and unfolded (qZ0) states. The approach we developed in Nelson & Grishin36 extended this model in effect to permit all orders, or complexities of the micro-ensemble topologies. The dominant contributions in proteins were found to originate from a simple class of scaling topologies (specically, one or more folded nuclei, each potentially decorated by loops and ends, joined together by open unfolded segments) so that the unfolded part of the system could still be described as a non-interacting soup of open segments and loops just as in the original model. Although this approach neglects the shape (size) of the nuclei and excluded volume effects from nuclei attached to open segments and ends, it is (i) correct in order of magnitude, (ii) leads to qualitative level improvements in the calculation of transition state structures for most (but not all) of the small to moderate size (X-ray) proteins in Galzitskaya & Finkelstein33 and Garbuzynskiy et al.34 (about 30% on average36) and (iii) identies intermediates (for example, the CheY misfolded intermediate) that are unavailable to the lower complexity models. It should be pointed out, however, that even this approach inhibits some kinetically important processes. An inherent constraint of the two-state model is that every pair of amino acids that are in contact in the crystal structure become cross-linked when they freeze into their folded states. Consequently, sub-domains that interpenetrate, or are otherwise strongly connected in the crystal structure are inhibited from folding independently (i.e. in parallel42). This situation can only be addressed by including an additional state per amino acid which substantially complicates the problem, but it occurs in perhaps one out of 20 or so

34. 35.

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42. 43.
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Edited by M. Levitt (Received 12 October 2005; received in revised form 10 February 2006; accepted 10 February 2006)