Biomolecular folding in vacuo

At the molecular level, life, as we know it, goes on in water. A liquid milieu is doubtless essential to provide rapid mobility to both the small molecules whose traffic is so essential to life and to the functioning macromolecules which act upon them. The omnipresence of water has exerted a profound influence on the evolution of biomolecular function, specificity, and stability. The self-assembly, or folding, of protein molecules in particular has been thought to involve water most intimately. Therefore, the observations reported by McLafferty and coworkers (1) of processes resembling the folding and unfolding of cytochrome c ions in vacuum immediately commands our attention and a careful examination of our views on the dominant forces involved in protein folding. While theorists have studied proteins in vacuo for some time (2), experimental protein chemistry of any kind in the vacuum may seem novel. At equilibrium, proteins have low vapor pressures; they are hardly "soluble" in the vacuum. Nevertheless a variety of techniques such as electrospray and laser deposition can inject a few nanomoles of protein into the gas phase. Thus, mass spectrometry is now routinely used to sequence biomolecules (3). The high sensitivity and specificity of mass spectrometry have also made it useful for characterizing the state of proteins taken from solution. Beautiful work by Dobson and collaborators (4) uses mass spectrometry to characterize various folding intermediates in solution by exploiting solution-phase proton exchange to label which residues are exposed to solvent. Studying the dynamics of what happens to a biomolecule once in the vacuum is a difficult matter, however, since there are so few molecules available. In the study of McLafferty and co-workers (1), chemical reactions and lasers come to the rescue; they characterize the conformations of the protein in vacuo using gas-phase hydrogen-deuterium exchange, a process stoichiometrically analogous to the very different chemistry used in solution. Again the number of exchangeable protons should give an idea of the openness of the protein conformation during gas-phase folding. Since the molecules are held for a long time in a Fourier transform mass spectrometer, McLafferty and co-workers also can use infrared lasers to heat the molecules. This apparently causes them to unfold in vacuo. Collisions with other gas molecules can change the charge on the protein ions, thereby allowing conformational changes. This is analogous to the way pH changes modulate folding in solution. In each case the high sensitivity of mass spectrometry allows the monitoring of very subtle changes in a very small number of molecules so that kinetic studies can be carried out. These studies indicate that gas-phase folding involves not just two states but several different populations of protein molecules that display varying degrees of proton exchangeability and, therefore, different degrees of openness. McLafferty and co-workers convincingly show both reversible and irreversible thermal transitions between these different conformational states. Whether these transitions involve the same states as the folding in solution is a question still worthy of discussion. The thermodynamic and kinetic stability and uniqueness of the collapsed structures all need to be considered. Historically various "forces" have been thought to be important in protein folding. Textbooks highlight three effects in discussing protein stability (5). These are hydrogen bonding (6), hydrophobic forces (7), and packing (8). Each of these plays a role in the architecture of fully folded proteins. The local secondary structures, a-helix and the P-sheet, are quite obvious features of proteins and are dominated by hydrogen bonds. Nearly equally prominent in protein architecture is the segregation of apolar hydrophobic and polar hydrophilic residues into the interior and surface of the protein, respectively. It is also quite apparent that large cavities are exceptional in proteins. Proteins are nearly as close packed as amino acid crystals, so the repulsive forces and steric constraints are very important. Both hydrogen bonding and the segregation of polar and apolar residues should be affected strongly by the presence or absence of water, since the water can provide alternative hydrogen bonds and solvate the charges on the polar residues. From the point of view of hydrogen bonding, the stability of the native protein in vacuo is not compromised. Without the alternative of hydrogen bonding to the solvent, the classical secondary structures should be even more thermodynamically stable. Indeed, their presence was predicted theoretically by Pauling without any concern for solvation phenomena. On the other hand, at first glance the contribution of hydrophobic forces to stability would seem to be a problem in vacuo. The vacuum being very apolar can be thought of as a very hydrophobic medium much like the lipid of a biomembrane. From this viewpoint it would seem sensible for a protein to turn inside out with its hydrophilic or polar residues inside and hydrophobic apolar residues outside. A transition to such a kind of collapsed structure cannot be ruled out so far by the experiment reported in this issue (1). It is worth bearing in mind, however, that the hydrophobic force is actually a composite of many microscopic forces. Microscopically one contribution to stability is the ordinary van der Waals attraction between the residues of the polypeptide. This is still present in vacuo. A separate contribution comes from van der Waals attractions between the water molecules and the protein atoms and between the waters themselves. Privalov has argued on the basis of microcalorimetry studies of solutionphase folding transitions that most of the stabilization, in fact, comes from the intramolecular van der Waals forces themselves and that the residual interactions with water are actually destabilizing (9). On this basis we would expect the folded protein structure to be at least metastable. This would be in accord with the many computer simulations of proteins in vacuo that usually hold together for a few nanoseconds of real time. Could the protein in its native solution structure be globally thermodynamically stable in vacuo; that is, are there no lower-energy conformations? The simple hydrophobic vacuum picture would argue otherwise! It is possible, however, that packing forces could provide all of the needed specificity. Richards (8), Chan and Dill (10), and many others to varying degrees have argued that many (but perhaps not all) structural features of folded proteins can be described in what statistical mechanicians would call a "van der Waals" picture. In this picture a relatively nonspecific attractive force causes the protein molecule to become compact. When the density is high enough, excluded volume and packing forces can then cause a specific ordered structure to arise, much like the crystallization of hard spheres