The amide III region of the peptide infrared and Raman spectra has been used to determine the relative populations of the three major backbone conformations (P II , β, and α R ) in 19 amino acid dipeptides. The results provide a benchmark for force field or other methods of predicting backbone conformations in flexible peptides. There are three resolvable backbone bands in the amide III region. The major population is either P II or β for all dipeptides except Gly, whereas the α R population is measurable but always minor (≤10%) for 18 dipeptides. (The Gly φ,ψ map is complex and so is the interpretation of the amide III bands of Gly.) There are substantial differences in the relative β and P II populations among the 19 dipeptides. The band frequencies have been assigned as P II , 1,317-1,306 cm −1 ; α R , 1,304-1,294 cm −1 ; and β, 1,294-1,270 cm −1 . The three bands were measured by both attenuated total reflection spectroscopy and by Raman spectroscopy. Consistent results, both for band frequency and relative population, were obtained by both spectroscopic methods. The β and P II bands were assigned from the dependence of the 3 JðH N ,H α Þ coupling constant (known for all 19 dipeptides) on the relative β population. The P II band assignment agrees with one made earlier from Raman optical activity data. The temperature dependences of the relative β and P II populations fit the standard model with Boltzmann-weighted energies for alanine and leucine between 30 and 60°C.vibrational spectroscopy | backbone conformations | spectral populations | aqueous solution A basic unsolved problem in protein folding is making accurate calculations of the folding energetics of flexible peptides. Accurate experimental results for the major backbone conformations are needed to test prediction methods. Even the simple problem of calculating the φ,ψ map of the alanine dipeptide is beyond the reach of standard force fields used in molecular dynamics simulations (1). There are two probable reasons: one is that the energy differences between the major backbone conformations are small, and the second is that standard force fields contain so many parameters that errors cannot be found readily by comparing simulations with experimental results. The ability to calculate accurately the relative energies of the various backbone conformations is needed for simulating early stages of the protein folding process, which is an important current problem in molecular biophysics.When simulated by standard force fields, the φ,ψ map of the alanine dipeptide has three major conformational basins, P II , β, and α R . Different force fields agree on this point but give widely varying results for the populations in the three basins (1). The φ,ψ maps of the 19 amino acid residues (Pro excluded) from the protein structure database show the same three basins and are similar in outline for the various residues if data for Gly and Pre-Pro are excluded in addition to Pro (2). The three basins are centered approximately at P II (−75°, 145°), β (−120°, 120°), a...

The preferences of amino acid residues for , backbone angles vary strikingly among the amino acids, as shown by the backbone angle found from the 3 J(H␣,HN) coupling constant for short peptides in water. New data for the 3 J(H␣,HN) values of blocked amino acids (dipeptides) are given here. Dipeptides exhibit the full range of coupling constants shown by longer peptides such as GGXGG and dipeptides present the simplest system for analyzing backbone preferences. The dipeptide coupling constants are surprisingly close to values computed from the coil library (conformations of residues not in helices and not in sheets). Published coupling constants for GGXGG peptides agree closely with dipeptide values for all nonpolar residues and for some polar residues but not for X ‫؍‬ D, N, T, and Y, which are probably affected by polar side chain-backbone interactions in GGXGG peptides. Thus, intrinsic backbone preferences are already determined at the dipeptide level and remain almost unchanged in GGXGG peptides and are strikingly similar in the coil library of conformations from protein structures. The simplest explanation for the backbone preferences is that backbone conformations are strongly affected by electrostatic dipole-dipole interactions in the peptide backbone and by screening of these interactions with water, which depends on nearby side chains. Strong backbone electrostatic interactions occur in dipeptides. This is shown by calculations both of backbone electrostatic energy for different conformers of the alanine dipeptide in the gas phase and by electrostatic solvation free energies of amino acid dipeptides.amino acid conformations ͉ dipeptides ͉ electrostatic screening T he origin of the differences among the intrinsic backbone preferences of the 20 aa is an unsolved puzzle, and these intrinsic preferences (1-6) are an important part of the local structure (7,8) in unfolded peptide chains that may be used to guide the folding process at early stages of folding. The ''intrinsic'' conformational preferences are specified by the backbone , angles found in short peptides and in the ''coil library'' (1-6) of Protein Data Bank residue conformations for residues outside repetitive secondary structures (helices, sheets). The existence of different backbone preferences in short peptides in water is demonstrated by values of the backbone angle , which can be measured by NMR from the 3 J(H ␣ ,H N ) coupling constant (vicinal coupling constant between C ␣ H and NH protons) by using the Karplus relation (9). These coupling constants are averaged over all backbone conformations that are present. Individual -values are obtained for residues in the coil library of the Protein Data Bank, and corresponding values of 3 J(H ␣ ,H N ) are found from the Karplus relation and then averaged. In the coil library of Smith et al. (3), average values of 3 J(H ␣ ,H N ) range from 5.9 Hz for Gly and 6.1 Hz for Ala to 7.7 Hz for Val. In addition to the different intrinsic backbone preferences, there is a substantial neighboring residue effect...

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

Hydrophobicity plays an important role in numerous physicochemical processes from the process of dissolution in water to protein folding, but its origin at the fundamental level is still unclear. The classical view of hydrophobic hydration is that, in the presence of a hydrophobic solute, water forms transient microscopic "icebergs" arising from strengthened water hydrogen bonding, but there is no experimental evidence for enhanced hydrogen bonding and/or icebergs in such solutions. Here, we have used the redshifts and line shapes of the isotopically decoupled IR oxygen-deuterium (O-D) stretching mode of HDO water near small purely hydrophobic solutes (methane, ethane, krypton, and xenon) to study hydrophobicity at the most fundamental level. We present unequivocal and model-free experimental proof for the presence of strengthened water hydrogen bonds near four hydrophobic solutes, matching those in ice and clathrates. The water molecules involved in the enhanced hydrogen bonds display extensive structural ordering resembling that in clathrates. The number of ice-like hydrogen bonds is 10-15 per methane molecule. Ab initio molecular dynamics simulations have confirmed that water molecules in the vicinity of methane form stronger, more numerous, and more tetrahedrally oriented hydrogen bonds than those in bulk water and that their mobility is restricted. We show the absence of intercalating water molecules that cause the electrostatic screening (shielding) of hydrogen bonds in bulk water as the critical element for the enhanced hydrogen bonding around a hydrophobic solute. Our results confirm the classical view of hydrophobic hydration.hydrophobic hydration | hydrogen bonding | IR spectroscopy | electrostatic screening | ab initio molecular dynamics D espite its great importance in numerous phenomena, the origin of hydrophobicity remains one of the most disputed topics in science (1-5). Experimental studies have shown that small purely hydrophobic solutes (alkanes and noble gases) in water increase the order (6, 7) and restrict the mobility (8) of neighboring water molecules. There are several opposing views on how to explain these data. The classical view is that a solute modifies water structure by forming transient, semiordered clathrate-like clusters ("icebergs"; used here only as a loose term) around it, arising from enhanced water hydrogen bonding (H bonding) (6, 7). This enhancement is brought about by either strengthening (9) or increasing the number of water to water H bonds (10). The classical view explains the characteristic changes in the thermodynamic variables of hydrophobic hydration (positive ΔG, ΔC p , negative ΔS, and ΔH) and the restricted mobility of water molecules observed by NMR (8). Neutron diffraction (11, 12) and extended X-ray absorption fine structure (EXAFS) (13) studies, however, show that the water molecules around small purely hydrophobic molecules do not differ significantly from those in pure liquid water. According to the dynamic view, the hydrophobic solute causes slowdown of t...