Faculty of mathematics and physics. University of Ljubljana PROTEIN FOLDING

Faculty of mathematics and physics University of Ljubljana PROTEIN FOLDING Author: Matjaž Zemljic Adviser: prof. dr. Dragan Mihailovic Ljubljana, november 2001 1

CONTENTS Abstract...2 1 Introduction...3 2 Proteins 2.1 Protein's role...3 2.2 Synthesis of proteins...3 3 Protein folding 3.1 Levels of protein's structure...5 3.2 Energy diagrams of proteins...6 3.3 Kinetics of folding...7 4 Folding experiments 4.1 General properties...8 4.2 Folding of apo-myoglobin...8 4.3 Experimental equipment...9 4.4 Vibrational characteristics of amide I band...10 4.5 Results...10 5 Conclusion...13 6 References...14 ABSTRACT Some general facts about proteins are presented in the beginning. Then I describe the structural stages adopted by a protein during the folding process. The energy landscape theory is introduced and the Levinthal s paradox is mentioned. Second part of this seminar is experimental. Here I describe a realistic experiment that involves a temperature-jump technique which seems to have a bright future in discovering fast folding processes. 2

1 INTRODUCTION The shape of a protein molecule is just as important as its chemical composition, and one of the challenges is to understand how a long one-dimensional molecule folds into a working three-dimensional structure. In the last decade physical scientists have made real progress in understanding how proteins fold. If we can understand the physical process of protein folding then we may be able to develop computer algorithms that can predict the structure of a protein from its amino acid sequence much more rapidly than experiments currently allow. Recently biochemical researchers have learned that several diseases are caused by defects in the protein folding process. The most notable of this is BSE(mad cow disease) which is caused by incorrectly folded proteins in the brains called prions. If protein folding process can be controlled, it may be possible to prevent similar brain disorders in humans and animals. 2 PROTEINS 2.1 Protein s role We do not exaggerate if we claim that proteins are the most important organic molecules within cells. Quick proof is that they represent more than half of the dry mass of the cell. Protein's role is dual: -they are structural units, which means that many parts of the cell are made of them(the membrane, the cytoskeleton...) -they enable or are involved in many processes taking place in the cell (such as catalysis of chemical reactions as enzymes, transport within and between cells is handled by them...) 2.2 Synthesis of proteins Proteins are biopolymers, which means that they consist of basic sub-units called amino acids. Amino acids are biomonomers made of hydrogen, oxygen, carbon and nitrogen. There are 20 different amino acids present in living beings differing from one another in a part called the residue(r on Fig.1), while other parts(amino and carboxyl group, alpha-carbon) are the same. Figure 1:Structure of an amino acid 3

Unfolded protein is a long chain molecule that contains from 50 to 1000 (300 on average) amino acids. Neighbouring amino acids in the chain are bonded together by peptide bonds formed between CO group of the first amino acid and NH group of the next. A molecule of water is released when the bond is formed(fig.2). Peptide bonds are covalent and therefore very strong. If we simplify we can say that organisms differ from each other because they consist of different proteins. As there are 20 candidates for each position in the chain, there are many possible sequences even for the shortest proteins assuring the variability of organisms. Figure 2:Forming of polypeptide chain by peptide bonds(co-nh) The sequence of amino acids in a protein is called the primary structure. Information about the primary structure of all proteins present in a certain organism is contained in a special molecule called the DNA molecule. Chapters about the genetic code(how the information about protein's primary structure is coded in the DNA) and synthesis of proteins(how the information in the DNA is translated to the sequence of amino acids) exceed this seminar and won't be explained. However the important output of those chapters is that at the end of the protein synthesis process we get an unfolded protein with defined primary structure which, as we shall see, contains all the important information about protein's life. As proteins are synthesised on the ribosome, they don't remain straight chains for a long time, because of the specific interactions between residues themselves and between residues and a solvent(water), which always surrounds proteins(cells mostly consist of water). The first process, which should be mentioned here, is hydrogen bonding between CO and NH groups of the amino acids. Furthermore some of the residues are hydrophobic which means that they rather associate with other residues 4

than with water and other are hydrophilic so like to interact with water. The hydrophobic\hydrophilic characteristics of an amino acid completely depend on the type of the residue. The hydrophilic interactions are polar or ionic and the hydrophobic interactions are non-polar. Because a chain of amino acids does not represent the stablest state of a protein interactions trigger the folding process. 3. PROTEIN FOLDING 3.1 Levels of protein's structure As we mentioned primary structure of a protein is determined by the sequence of the amino acids. Next level of organisation is called the secondary structure. Protein adopts this structure, when the chain forms localised shapes known as helices and sheets, which are formed as a consequence of hydrogen bonding between nonneighbouring amino acids. We know many types of sheets. The tertiary structure is formed because different interactions between helices and\or sheets occur(hydrogen, ionic and covalent bonds). Hydrophobic residues form an interior of the protein(protein's core), while hydrophilic residues are present on the surface. This is also the final structure for smaller proteins. Figure 2: Levels of protein s structure 5

The quaternary structure is a characteristic of bigger proteins made from several already tertiary folded chains. Some additional contacts(mostly van der Waals) are made between them to form a compact protein. 3.2 Energy diagrams of proteins When the protein chain is synthesised on the ribosome it can not immediately perform its biological function. Protein is biologically active when it adopts a unique threedimensional structure. The process, which leads to protein s final shape, is called protein folding. We must say that this process has not been fully understood yet, but some general facts have been accepted by scientists. The system, which is our current interest, is a solution consisting of water and proteins, which all have the same primary structure. It is known that a protein, which N 1 consists of N amino acids, can adopt γ conformations or 3D structures, where γ is the finite number of possible orientations between neighbouring amino acids[2]. So each three-dimensional structure of a protein at the end of folding is exactly determined by the primary structure and all orientations between neighbouring amino acids in the folded structure. The angles between amino acids are the independent variables of the conformational space. As there are different interactions(meaning quantitatively) present in each conformation we can calculate the energy of a protein as a function of parameters which define each conformation(angles between amino acids). Energy of conformations does depend on the type of the solvent because of the specific interactions between residues and the environment, but we won't treat it as a variable, because this would complicate our discussion. The theory of calculating conformational s energy is called the energy landscape theory. Instead of calculations, I am going to present a general fact which is produced from this theory. It says that we can split proteins into two groups: good and poor folders[1]. A good folder's energy diagram has one deep global minimum(fig(3a, 3b, 3c)). A state corresponding to this minimum is called the native state. When the temperature is far below Tf each protein can adopt only conformations that are very close to native (Fig(3c)). As we pump the energy to our system which is indicated by an increase of temperature in the surrounding water, the number of possible conformations that can be occupied increases. What can not be seen on Figure(3) is that the energy levels are discrete. The line on (Fig(3b)) represents the upper limit of accessible states. Because a A*kT(A is a constant) of the thermal energy is loaded in each energy freedom level of such a protein, all atoms in the protein vibrate. If this vibrations are strong enough(when T is high enough) this deforms a protein and causes transitions between states in the conformational space which are separated less than A*kT by each other. So under each condition we can not say that protein has adopted a single conformation. In opposite it runs through the allowed part of the conformational space all the time, because of the thermal energy loaded in vibrational levels of a protein. On the other hand poor folder's energy diagram consists of many local shallow minimums. This means that at temperatures far below Tg a single protein can adopt conformations in the neighbourhood of an each state defined by one of the minimums (Fig(3f)). But we have a lot of proteins with the same primary structure that are being synthesised in a cell at a certain moment and they randomly choose a local minimum to adopt. So even at low temperatures we get proteins which are running through the allowed conformational space but almost each of them around its own local minimum. 6

Figure 3: Energy of an amino acid chain as a function of a coordinate that describes every 3D conformation it can adopt This means that we don t have a single favourable state. As the energy is pumped to the system the number of allowed conformations increases (Fig(3e)). This two types of diagrams represent the extreme possibilities. The energy diagrams mostly consist of some relatively deep minimums and so define more stabile states of a protein. It is known that random chosen primary structures(artificially made proteins in labs) are poor folders. On the other hand all proteins found in living beings are good folders, because the process of evolution has eliminated the poor ones[1]. So the question is why would nature favour good folders. At temperatures far below Tf or Tg(this are the temperatures in living organisms) we saw that the allowed conformational space for a good folder is very narrow meaning that a protein can adopt only few conformations. Although the protein keeps running through the allowed part of the conformational space the time probability to find a protein in native state is very high. And native state is the only biologically active state capable of completing the job for which the protein synthesis was started in the beginning. So it is a favourable state. In opposite poor folder can reach each of the many minimums at the end of folding. Of course one of the minimums does represent a biologically active state but only few of the synthesised proteins get trapped into it. So the loss of energy when synthesising a poor folder is too big. The stability of states in shallow minimums is also weaker because small thermal excitations can cause dislocation of a protein from a certain state. 3.3 Kinetics of folding Until now we were examining proteins in a solution under equilibrium conditions (temperature, pressure ) which as we saw don t mean an equilibrium state for a single observed protein. 7

Now we are also interested in the kinetics of a protein, in other words what is the way from an unfolded chain to the final state. This is the biggest unsolved chapter in N 1 protein folding examination. As we mentioned above, γ is the number of possible conformations for a given sequence of N amino acids. Even for a small protein of 101 amino acids(γ=4) there are 100 4 possible conformations(for large proteins γ=2). If we assume that a protein hunts randomly for its final conformation at a rate of about searches per second, it would take longer than the age of the Universe for a single protein to fold up. As it is known from the experiments proteins can completely fold in seconds or minutes. This is known as the Levinthal's paradox[2]. Consequently many theoretical and numerical approaches reduce the number of possible conformations by grouping smaller parts of the proteins together and presuming that the dynamics are somehow guided. So the protein is split into a number of smaller components with quasi-independent dynamics. Within this components the correct final contacts are made. After that contacts on higher level between those components are formed. 13 10 4. FOLDING EXPERIMENTS 4.3 General properties We are examining a good folder. At the end of the folding process protein runs through the allowed part of the conformational space whose size is determined by temperature as we saw on Figure (3b,3c). This is true only for reasonable temperatures. If we heat the solution above approximately 50 C most of the proteins would unfold or denature (Fig(3a,3d)), so tertiary and secondary contacts would be broken. Denaturation of proteins can also be achieved by some other methods, like adding chemical denaturants to the solution or by extreme values of ph. Moreover, when we reverse the denaturing conditions(the initial conditions are restored) it turns out that proteins can spontaneously refold. What is important is that the refolded protein s conformational space is the same as before unfolding or if we simplify: protein is biologically active again. The conclusion is that the information about all the possible three-dimensional structures of a protein is contained in the primary structure which isn t destroyed by unfolding. This statement is known as the second genetic code [2]. Unfolding-refolding process seems to be reversible(for thermal unfolding up to 70 C). So to study protein folding, a protein must be first unfolded by using appropriate methods. We know that the shortest folding times for smallest proteins are tens of microseconds and for bigger proteins seconds or minutes. But if we want to examine the folding processes of intermediate structures(such as the folding times for forming α helices, β hairpins or tertiary contacts) we should know that this takes only some nano- or even picoseconds. If we reverse denaturing conditions to trigger folding by diluting the solution with mixing, that typically takes from 100µs to 1ms(the dead time). The dead time is the time needed that the equilibrium conditions are restored in the solution, because only then our detection of changes in protein's structure is relevant. Detection within the dead time may also involve changes in solvent's properties. Obviously, some submillisecond processes are already finished within this time. The challenge of resolving the fast processes has motivated many physical 8

scientists to develop new methods to study folding on the submillisecond scale. So new techniques have been developed. One of them, which I am going to present in this seminar is called a laser induced temperature jump technique. It provides temperature jumps of typically 15-30 in 10 ns for a sample volume of approximately 10 3 mm 3. 4.2 Folding of apo-myoglobin Myoglobin is a protein which preserves oxygen in the muscle cells. It contains eight strands of mostly α helical segments labelled A-H on the Figure(4) and a heme group, where the iron ion is present, which bands the oxygen. The sample in our experiment is a protein called apo myoglobin in D2O, which doesn t have the heme group. We remove it because it disturbs the infra-red spectroscopy. Due to its simple structure apo myoglobin is a prime candidate for the study of protein folding. Figure 4:Structure of apo myoglobin 4.3 Experimental equipment The general approach to the rapid initiation and characterisation of folding reactions is to use temperature jump to unfold a protein and to monitor the resulting changes using infrared spectroscopy. The temperature jump is achieved using a pump laser pulse generated from the Nd- YAG pumped dye laser and the Nd-YAG fundamental laser. The FWHM of the pulse is 10 ns and it carries energy of 3mJ which can be varied by changing the laser s power. This allows us to control the height of the T-jump. The pulse passes through LiNbO3 crystal, which changes its wavelength to 1.9µm. This wavelength corresponds to the peak of a weak D2O IR absorption band and is ideal for our purposes, because most proteins do not absorb near 2µm. This is important because we don't want to affect proteins in any other way than by heating the surrounding. So the laser energy is absorbed by water, resulting in a maximum T-jump of 20 in approximately 10 ns(the dead time).we should mention that proteins are also heated within the dead time. About 90% of the energy is transmitted through 100µm, which is the sample cell length. The high transmission ensures a nearly uniform temperature profile in the 9

sample cell. The diffusion of heat out of the interaction volume takes several milliseconds, so the temperature remains nearly constant up to 1 ms. Equilibrium or time-independent characteristics of the solution are determined by measuring the absorption spectra of the infrared beam, which passes through the sample. This is used to measure the size of the T-jump and to detect properties of the sample in equilibrium. The frequency range of the beam is from 1550 to 1750 cm 1. A lead-salt IR diode laser is used as a source of a probe IR pulse. This pulse is monochromatic, because it passes through a filter positioned before the sample, and used to measure properties of the sample changing in time. The FWHM of the probe pulse is around 100 ps. Changes in the transmission of the IR pulse through the sample are detected by a HgCdTe detector. The detector s response time is approximately 10 ns. We use a split sample cell, one compartment containing D2O and polypeptides and the other containing only D2O. Parallel to measuring the spectra of the solution, we also measure the spectra of D2O. Than we subtract it from the solution s spectra and obtain only the changes caused by polypeptide s properties. Figure 5: The nanosecond temperature-jump apparatus 4.4 Vibrational characteristics of amide I band As we know each molecule has some characteristic eigenfrequencies, which can be calculated, assuming that the potential between atoms is harmonic for small excitations. If we agitate such a molecule with light having the right frequency in its spectra, it would be absorbed by the molecule. The sensitivity of such vibrational spectroscopy to protein structure is welldocumented. The amide I band, which is present in apo myoglobin, consisting primarily from the C=O groups(fig(2)), is a particularly good indicator of structural changes of a protein, because of its sensitivity to hydrogen bonding. As we know hydrogen bonds are responsible for secondary and tertiary contacts. The amide I band as a probe is not a specific region in apo myoglobin. The eigenfrequencies are the average effect of the C=O bonds in the whole protein. Previous studies have shown that in D2O unfolded structures generally exhibit amide I frequencies between 1665-10

1675 cm 1. Characteristic frequencies for native structure are from 1650-1655 cm 1. There have also been some studies, which revealed that frequencies for secondary contacts made in the protein are in the interval between 1630-1645 cm 1 [3]. We have to mention, that some of the proteins have adopted only secondary formations in our specifical case. 4.5 Results Figure(6a) shows the absorbance spectra of apo myoglobin in D2O as a function of temperature from 32-55 C. We obtain different T-jumps by changing the laser s power. The D2O background has been subtracted from each spectra. Figure 6: Equilibrium curves showing thermal unfolding of apo myoglobin We used equilibrium IR spectroscopy here because the solution is heated in 10 ns. Later we ll see that proteins are unfolded in 150µs so we have an equilibrium for about 0.8ms. As mentioned temperature starts to decrease after 1ms. Due to the strong overlap of the subcomponents Figure(6a) doesn t show any dramatic changes which in opposite can be seen in the difference spectra on Figure(6c), where spectra at 32.8 C has been subtracted from other ones. As the temperature is increased the signal at frequencies characteristic for secondary and native structures(1630-1655 cm 1 ) is 11

decreased, while the unfolded signal at 1673 cm 1 is increased. This is the proof that apo myoglobin is thermally unfolded. Figure(7) also shows thermal unfolding of apo myoglobin monitored at characteristic frequencies of amide I band. We can see a dramatic change of signal height for native structure around 59 C, which closely resembles the melting temperature obtained from another commonly used method called circular dichroism. In contrast the thermal denaturation of the secondary structure doesn t show a sharp transition with temperature, instead exhibiting a continuous loss of signal over the entire temperature range. This equilibrium IR data shows that unfolding of native apo myoglobin and secondary structure are independent processes, because they follow entirely different curves. There appears to be no influence of the unfolding of the native apomg on the unfolding of the secondary structure, because native proteins don t cause an increase of the secondary signal when they unfold. Consequently the signal at 1676 cm 1 constantly increases due to unfolding of the secondary structures and shows a high increase at 59 C. Figure 7: Thermal unfolding of apo myoglobin measured at characteristic frequencies for different structures The next measurement is time dependent and we have to use IR probe pulses instead of beam to detect processes on nanosecond scale. Unfolding of apomg is initiated by laser pump pulse, which causes the T-jump for 15 from initial 45 C to 60 C and the kinetics are followed in the amide I region. The lead-salt IR laser shoots a pulse of 12

length of 10 nanoseconds through the sample. After that the pulse is detected by a MgCdTe detector. Each point on Figure(8) is obtained by changing the time delay between probe and pump pulse in a step of 5 ns which allows us to follow changes in the protein s structure. Figure (8) shows the tree vibrational frequencies, which correspond to changes in secondary structure(1632 cm 1 ), native structure(1655 cm 1 ) and unfolded structure(1664 cm 1 ). The background D2O response has been subtracted from these data. Points at 5 and 10ns are irrelevant because they are measured within the dead time of the method. Two clearly resolved kinetic phases, separated by several orders of magnitude, can be observed. The transient at 1632 cm 1 is dominated by a single fast kinetics phase with a relaxation time of 48 ns, while the transient at 1655 cm 1 is characterised by a slower phase with a relaxation time of 132 µs. The early time spectrum has a minimum at 1632 cm 1, which is indicating loss of secondary structures, while longer time spectrum is identical to the equilibrium difference spectra seen on Figure(6), with the major bleach positioned at the native frequency. All three signals begin to get closer to the zero line, on the abovemillisecond scale, because the heat diffuses out of the interaction volume and the spectra returns to the equilibrium spectra at initial 45 C. The time constant for heat diffusion is approximately 7 ms. That can t be really seen on Figure(8) because the time range is too short. Figure 8: Time dependent characteristics of absorption for different structures of apo myoglobin From the time dependant measurements one can see that unfolding kinetics can be t / τ perfectly described by a single exponential function of a form A=A0*exp where A is a probe used in experiment(in our case the absorption intensity)[1]. If we assume the reversibility of folding-unfolding process the folding kinetics can be described by t / τ a function of form A=A0*(1- exp ). The result of our experiment is that unfolding time for apomg is 132 µs and unfolding time for secondary structured apomg is 48 ns. 13

5. Conclusion The problem of protein folding is not in studying long time processes like the whole folding of a protein from a chain to the final structure. This can be easily followed by methods like ultrafast mixing. What is in interest today is an examination of the early stages of folding and short-time stages during folding. This processes are happening on the time scales of pico- or even femtoseconds. Only the laser T-jump approaches seem to be useful here due to very short dead time interval. So this techniques need to be improved. Does the investigation of how natural proteins fold answer the practical questions of structure prediction and misfolding-disease prevention? To predict structure requires large scale computer simulations and the problem is that we do not know if the energy functions are good enough to be used properly to fold a protein in a realistic way. As for misfolding diseases, little practical has yet come from physics and chemistry. But deeper knowledge of protein-folding may allow the design of drugs that will bind to specific protein conformations, thereby altering the energy landscape and preventing misfolding. 6. References (1) Peter G. Wolynes, William A. Eaton ; The physics of protein folding ; PHYS WORLD 12: (9) 39-44 SEP 1999 (2) Robert Challender, Rudolf Gilmanshin, Brian Dyer, William Woodruff ; Protein physics ; PHYS WORLD 7: (8) 41-45 AUG 1994 (3) Rudolf Gilmanshin, Skip Williams, Robert H. Challender, William H. Woodruff, Brian R. Dyer ; Fast events in protein folding:relaxation dynamics of secondary and tertiary structure in native apo myoglobin ; P NATL ACAD SCI USA 94: (8) 3709-3713 APR 15 1997 14