Top Curr Chem (2015) 364: 225–270 DOI: 10.1007/128_2014_580 # Springer International Publishing Switzerland 2014 Published online: 25 March 2015
Isolated Neutral Peptides Eric Gloaguen and Michel Mons
Abstract This chapter examines the structural characterisation of isolated neutral amino-acids and peptides. After a presentation of the experimental and theoretical state-of-the-art in the field, a review of the major structures and shaping interactions is presented. Special focus is made on conformationally-resolved studies which enable one to go beyond simple structural characterisation; probing flexibility and excited-state photophysics are given as examples of promising future directions. Keywords Amide • Amino-acid • Backbone-side chain interactions • Conformation-selective IR spectroscopy • Gas phase laser spectroscopy • Hydrates • Secondary structures • Supersonic expansion
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Two Synergetic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Lessons from the Confrontation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Peptide Structures Identified in the Gas Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Secondary Structures of Protein Backbones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Backbone-Side Chain Interactions in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Side Chain–Side Chain Interactions in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
226 229 229 233 236 239 239 243 245
E. Gloaguen (*) CNRS, INC and INP, Lab. Francis Perrin, 91191 Gif-sur-Yvette, France CEA, IRAMIS, Laboratoire Interactions, Dynamique et Lasers, 91191 Gif-sur-Yvette, France e-mail: [email protected] M. Mons (*) CEA, IRAMIS, Laboratoire Interactions, Dynamique et Lasers, 91191 Gif-sur-Yvette, France CNRS, INC and INP, Lab. Francis Perrin, 91191 Gif-sur-Yvette, France e-mail: [email protected]
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3.4 Terminal-Controlled Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Microsolvation Structures and Complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Beyond Short Linear α-Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Pushing Gas Phase Investigation to Its Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Added Value of Conformation-Resolved Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Flexibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Excited States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
245 246 247 247 248 248 255 258 259
Abbreviations Ac Aib BB Bn CI CT DFT FC FEL FTIR IR LE Me NCI OPO PES SC SEP UV VUV Z
Acetyl Aminoisobutyric acid Backbone Benzyl Conical intersection Charge transfer Density functional theory Franck–Condon Free electron laser Fourier transform infrared Infrared Locally excited Methyl Non-covalent interactions Optical parametric oscillator Potential energy surface Side-chain Stimulated emission pumping Ultraviolet Vacuum ultraviolet Benzyloxycarbonyl
1 Introduction Peptides and proteins are complex systems, whose functionalities in the living world ensue from a subtle compromise between structuration and flexibility [1]. On one hand, their backbone has its own steric limitations caused by the rigidity of the peptide bonds which link residues together, restricting the set of conformations and therefore the types of possible secondary structures [2, 3]. On the other hand, the large diversity brought by the side chains of the 20 natural amino-acid residues explains the tremendous variety of tertiary and quaternary
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structures, together with the various tasks performed by these molecules, including enzymatic properties, molecular recognition, mechanical tasks, etc. Besides the classical techniques for structural determination of proteins, namely X-ray diffraction or nuclear magnetic resonance, molecular modelling has become a complementary approach, providing refined structural details [4–7]. This view on the atomic scale paves the way to a comprehensive study of the correlations between protein structure and function, but a realistic description relies strongly on the performance of the theoretical tools. Nowadays, a full size protein is treated by force fields models [7–10], and smaller motifs, such as an active site of an enzyme, by multiscale approaches involving both quantum chemistry methods for local description, and molecular mechanics for its environment [11]. However, none of these methods are ab initio: force fields require a parameterisation based on experimental data of model systems; DFT quantum methods need to be assessed by comparison against high level ab initio calculations on small systems. In such a context, isolated model systems can play the role of benchmarks, on which theory and experiment can be confronted at a very high level of detail. At the turn of the century, this rationale has led gas phase spectroscopists to devote experimental effort towards simple systems of biological relevance [12, 13]. Small building blocks of proteins were first targeted: conformational distributions of these flexible systems, as well as the interactions that shape molecules, in particular their H-bonding networks, have been the focus of these pioneering studies [14–20]. In this spirit, the classical gas-phase methods of the physicist, inherited from atomic or small molecule spectroscopy, namely optical absorption, either in the microwave, IR/Raman or UV spectral range, have been mobilised to tackle these issues. First, microwave experiments, carried out in supersonic expansions, remain devoted to relatively small molecules, from models of biomolecules to more recently investigated natural and capped amino-acids and sugars [21]. The advantage of this approach stems from its direct sensitivity to the structure, through the rotational constant, allowing it to provide stringent tests to the theoretical structures calculated. However, similar to X-ray crystallography or NMR studies, these experiments remain only indirectly sensitive to the intramolecular interactions which shape the molecule. In contrast, vibrational probes, which are more directly sensitive to their immediate environment, provide an invaluable opportunity to document the interactions occurring in these systems. In particular, NH, OH, and CO stretching modes appeared to be promising diagnostics, leading to an accurate H-bond network characterisation, and have therefore been explored by several groups. Although Raman absorption [22–25], FTIR [26–31] or IR/VUV [32, 33] spectra can be successfully carried out in an expansion, these techniques suffer from their lack of conformer selectivity. Conversely, the selectivity of double resonance IR/UV techniques has raised considerable interest, leading laser spectroscopists to provide an accurate picture of the structure of each conformer through the IR spectra, as well as information on the conformational populations through UV spectroscopy. This latter approach has proved to be quite successful [34–39]. It has been applied to numerous biosystems, either neutral or charged, isolated or in small clusters. Owing to the extent of this field, the present review focuses on globally neutral peptides, isolated or microsolvated [40, 41].
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Characterisation of isolated neutral peptides through their vibrational signature resulted from the synergy between two complementary approaches: – Gas phase spectroscopy of jet-cooled neutral molecules benefited from the development of laser-vaporisation and laser-desorption techniques as well as their coupling with a supersonic expansion. Improvements of spectroscopic procedures involving several lasers, e.g. the IR/UV double resonance spectroscopy, helped to collect information on weakly populated conformers. – Quantum chemistry methods have been constantly improved, gaining in accuracy and transferability. Helped by the computer “Moore’s law” during the past two decades, theoretical studies have been able to treat larger and larger species at an increasingly better level of theory. The first studies focused on the identification of small species by comparison between their theoretical and experimental vibrational fingerprints. Starting from aromatic natural amino acids which possess a convenient UV chromophore (see Sect. 2), many seminal studies have been devoted to these natural model peptides. Their biological relevance can be questioned, in particular because the N and C natural terminals adopt a zwitterionic structure under physiological conditions which is usually not the structure observed in the gas phase. These natural peptides nevertheless constitute handy model systems on which experimental techniques and theoretical methodologies have been refined. This approach became increasingly difficult as the targeted systems were larger and larger. Among these, capped peptides, whose N and C terminals were made of complete peptide bonds, enabled progress to be made towards greater biological relevance. Successful investigation of systems as large as tripeptides stemmed from a gradual knowledge of species of increasing size, following the so-called bottom-up approach. A set of essential secondary structures has been identified, demonstrating the biological relevance of such gas-phase studies. By demonstrating that relevant models mimicking the backbone features of proteins could be studied in the gas phase at an unprecedented level of accuracy, these pioneering works opened new horizons and stimulated a large community. Taking advantage of decisive quantum chemistry progress, a more thorough approach appeared where a global theoretical study of the PES was conducted. Close comparison between the experimentally observed landscape and its theoretical counterpart provides much richer outcomes, allowing a fruitful interplay between experiment and theory. A comprehensive and precise PES enables a reliable assignment of the species observed, including all minor conformers. In return, failures of quantum chemistry methods to reproduce experimental observations could be spotted, guiding further theoretical developments. The aim of this review is first to describe the parallel advancements in both experimental and theoretical methods and procedures, together with the crossfertilisation that resulted. Examples of striking secondary structures of proteins isolated in the gas phase are given. Highlights on specific shaping interactions, including intermolecular interactions, e.g. with the solvent, provide an in-depth understanding of protein structure. In a last part, it is shown how conformationselective spectroscopy is able to document the diversity of the PES landscape of
Isolated Neutral Peptides
229
these species, thus revealing their flexibility. It is also shown how coupling IR and UV information enables spectroscopists to target conformer-selective photophysics in peptides. Finally, future prospects of gas phase conformation-selective spectroscopy are presented.
2 Two Synergetic Approaches 2.1
Experiment
Vibrational spectroscopy, based on IR or Raman absorption, is an efficient technique to identify the conformational structure of small molecules, including model peptide systems [22–33]. It is, however, confronted by spectral congestion when dealing with molecules of increasing flexibility with a pool of conformations which interconvert at room temperature. Advanced procedures are required to gain valuable conformation-specific spectroscopic information. An efficient cooling usually provided by a supersonic expansion is needed to remove the room temperature internal energy, typically 3.103 cm1 in a capped amino-acid. In addition, techniques such as temperature- or concentration-dependence studies are needed to identify the contributions of conformers and clusters [23, 27, 28, 30]. Several conformer-selective experiments were proposed, such as those based on a strong field deflection [42]. In this context, IR/UV double resonance experiments [43, 44] became quite popular. When conformer-selective excitation of a UV chromophore is possible, these experiments provide spectroscopists with an elegant way to record conformer-selective IR spectra [45]. Combined with classical thermal vaporisation techniques [46, 47] or with more sophisticated laser-desorption-based techniques [48–50] coupled to a supersonic expansion, IR/UV experiments have dramatically boosted the research field pioneered by Levy’s group [48]. They made possible the observation of vibrationally resolved IR spectra (Fig. 1), first from the NH stretch spectroscopy permitted by the development of table-top OPOs in this range [45]. From the basic principles of IR spectroscopy, H-bonding in jet-cooled peptides can be reliably identified, providing a first basis to conformational assignment, further refined by comparison with quantum chemistry calculations. Rapidly, several conformers of the three natural amino-acids Phe [16], Trp [20] and Tyr [56], and sometimes their hydrates [57, 58], were identified through their vibrational fingerprint, refining former pioneering studies based on UV spectroscopy only [48, 59–62]. Numerous studies on peptides containing one of the three natural aromatic residues followed [46, 47, 51–55, 63–118], the presence of a near-UV chromophore being the tribute to pay to gain the conformer-selectivity brought by the IR/UV double resonance technique. Non-natural residues derived from the natural UV chromophores have also been widely employed [52, 111, 119– 129]. Beyond this constraining aspect, the UV frequency and FC pattern being
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sensitive to the interactions involving the aromatic ring, they can provide very useful structural clues (Sect. 4.2). Of course, the principle of the experiment relies on the conformational selectivity of the UV chromophore. This is made possible by the intrinsic sensitivity of the UV transition to the chromophore environment, the cooling achieved in the expansion enabling the detection of frequency differences as small as a few cm1, in practice limited by the rotational contour of the UV bands. This selectivity can in general be achieved for small species, apart from spectral coincidence cases, which require a more sophisticated treatment [130]. In larger species, the spectral broadening caused by the difficulty in evacuating the internal energy results in a lack of UV resolution which affects the conformational selectivity [54, 89, 107, 108]. In these cases, one has to rely on the interpretation of the IR spectrum to figure out whether conformational selectivity is achieved or whether several species are simultaneously present. It should be noted that sequences deprived of UV chromophore can also been investigated by introducing so-called UV-tags which can be covalently [105, 107, 127, 131–145] or non-covalently bound, as in peptide-toluene complexes (Gloaguen E, Mons M, unpublished results). The assumption that the desired UV chromophore (often a phenyl ring) is weakly interacting with the rest of the molecule turned out to be a relatively seldom situation, owing to the huge flexibility of the peptides. Biases are introduced by what can be seen as parasitic interactions, e.g. NH-π [131], which prevent to extract any intrinsic data about the untagged system, especially in terms of conformational energetics. Gas phase peptides of various sizes have been investigated in several spectral ranges (Fig. 1a–e), the first of them, in both importance (number of studies), reliability and interpretative potentialities is probably the so-called amide A NH-stretch region (Fig. 1c, d). The NH stretch oscillator exhibits an exquisite sensitivity to its environment, and is virtually uncoupled with the potentially numerous other NH groups of the molecules. Then, each individual IR band bears qualitative information about the strength of the H-bond in which the corresponding NH group is engaged [34], apart from very specific cases where strong hyperconjugation effects involve the σ* orbital of the vibrational probe [146]. Moreover, other vibrational probes lie in the amide A NH-stretch region, and contribute to make this spectral range very popular: amine NH and carboxylic acid OH for natural peptides, indole NH fot Trp, imidazole NH for His, phenol OH for Tyr, alcohol OH for Ser and Thr as well as water OH in microhydrated peptides. For the simplest systems, the H-bonding content can be deduced just by “reading” the conformer-selective amide A spectrum. Spectroscopic tricks, such as spectral correlations between physically coupled NH oscillators in NH2-capped C-terminal groups [34, 70], together with the analysis of UV spectroscopic features (see Sect. 4.2), provide additional structural clues which have enabled the tackling of more complex species. Phe-containing dipeptides have been confidently assigned without relying on quantum calculations, and a ranking of the H-bonds according to their strength has been proposed (C5
Isolated Neutral Peptides Eric Gloaguen and Michel Mons
Abstract This chapter examines the structural characterisation of isolated neutral amino-acids and peptides. After a presentation of the experimental and theoretical state-of-the-art in the field, a review of the major structures and shaping interactions is presented. Special focus is made on conformationally-resolved studies which enable one to go beyond simple structural characterisation; probing flexibility and excited-state photophysics are given as examples of promising future directions. Keywords Amide • Amino-acid • Backbone-side chain interactions • Conformation-selective IR spectroscopy • Gas phase laser spectroscopy • Hydrates • Secondary structures • Supersonic expansion
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Two Synergetic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Lessons from the Confrontation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Peptide Structures Identified in the Gas Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Secondary Structures of Protein Backbones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Backbone-Side Chain Interactions in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Side Chain–Side Chain Interactions in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
226 229 229 233 236 239 239 243 245
E. Gloaguen (*) CNRS, INC and INP, Lab. Francis Perrin, 91191 Gif-sur-Yvette, France CEA, IRAMIS, Laboratoire Interactions, Dynamique et Lasers, 91191 Gif-sur-Yvette, France e-mail: [email protected] M. Mons (*) CEA, IRAMIS, Laboratoire Interactions, Dynamique et Lasers, 91191 Gif-sur-Yvette, France CNRS, INC and INP, Lab. Francis Perrin, 91191 Gif-sur-Yvette, France e-mail: [email protected]
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3.4 Terminal-Controlled Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Microsolvation Structures and Complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Beyond Short Linear α-Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Pushing Gas Phase Investigation to Its Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Added Value of Conformation-Resolved Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Flexibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Excited States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
245 246 247 247 248 248 255 258 259
Abbreviations Ac Aib BB Bn CI CT DFT FC FEL FTIR IR LE Me NCI OPO PES SC SEP UV VUV Z
Acetyl Aminoisobutyric acid Backbone Benzyl Conical intersection Charge transfer Density functional theory Franck–Condon Free electron laser Fourier transform infrared Infrared Locally excited Methyl Non-covalent interactions Optical parametric oscillator Potential energy surface Side-chain Stimulated emission pumping Ultraviolet Vacuum ultraviolet Benzyloxycarbonyl
1 Introduction Peptides and proteins are complex systems, whose functionalities in the living world ensue from a subtle compromise between structuration and flexibility [1]. On one hand, their backbone has its own steric limitations caused by the rigidity of the peptide bonds which link residues together, restricting the set of conformations and therefore the types of possible secondary structures [2, 3]. On the other hand, the large diversity brought by the side chains of the 20 natural amino-acid residues explains the tremendous variety of tertiary and quaternary
Isolated Neutral Peptides
227
structures, together with the various tasks performed by these molecules, including enzymatic properties, molecular recognition, mechanical tasks, etc. Besides the classical techniques for structural determination of proteins, namely X-ray diffraction or nuclear magnetic resonance, molecular modelling has become a complementary approach, providing refined structural details [4–7]. This view on the atomic scale paves the way to a comprehensive study of the correlations between protein structure and function, but a realistic description relies strongly on the performance of the theoretical tools. Nowadays, a full size protein is treated by force fields models [7–10], and smaller motifs, such as an active site of an enzyme, by multiscale approaches involving both quantum chemistry methods for local description, and molecular mechanics for its environment [11]. However, none of these methods are ab initio: force fields require a parameterisation based on experimental data of model systems; DFT quantum methods need to be assessed by comparison against high level ab initio calculations on small systems. In such a context, isolated model systems can play the role of benchmarks, on which theory and experiment can be confronted at a very high level of detail. At the turn of the century, this rationale has led gas phase spectroscopists to devote experimental effort towards simple systems of biological relevance [12, 13]. Small building blocks of proteins were first targeted: conformational distributions of these flexible systems, as well as the interactions that shape molecules, in particular their H-bonding networks, have been the focus of these pioneering studies [14–20]. In this spirit, the classical gas-phase methods of the physicist, inherited from atomic or small molecule spectroscopy, namely optical absorption, either in the microwave, IR/Raman or UV spectral range, have been mobilised to tackle these issues. First, microwave experiments, carried out in supersonic expansions, remain devoted to relatively small molecules, from models of biomolecules to more recently investigated natural and capped amino-acids and sugars [21]. The advantage of this approach stems from its direct sensitivity to the structure, through the rotational constant, allowing it to provide stringent tests to the theoretical structures calculated. However, similar to X-ray crystallography or NMR studies, these experiments remain only indirectly sensitive to the intramolecular interactions which shape the molecule. In contrast, vibrational probes, which are more directly sensitive to their immediate environment, provide an invaluable opportunity to document the interactions occurring in these systems. In particular, NH, OH, and CO stretching modes appeared to be promising diagnostics, leading to an accurate H-bond network characterisation, and have therefore been explored by several groups. Although Raman absorption [22–25], FTIR [26–31] or IR/VUV [32, 33] spectra can be successfully carried out in an expansion, these techniques suffer from their lack of conformer selectivity. Conversely, the selectivity of double resonance IR/UV techniques has raised considerable interest, leading laser spectroscopists to provide an accurate picture of the structure of each conformer through the IR spectra, as well as information on the conformational populations through UV spectroscopy. This latter approach has proved to be quite successful [34–39]. It has been applied to numerous biosystems, either neutral or charged, isolated or in small clusters. Owing to the extent of this field, the present review focuses on globally neutral peptides, isolated or microsolvated [40, 41].
228
E. Gloaguen and M. Mons
Characterisation of isolated neutral peptides through their vibrational signature resulted from the synergy between two complementary approaches: – Gas phase spectroscopy of jet-cooled neutral molecules benefited from the development of laser-vaporisation and laser-desorption techniques as well as their coupling with a supersonic expansion. Improvements of spectroscopic procedures involving several lasers, e.g. the IR/UV double resonance spectroscopy, helped to collect information on weakly populated conformers. – Quantum chemistry methods have been constantly improved, gaining in accuracy and transferability. Helped by the computer “Moore’s law” during the past two decades, theoretical studies have been able to treat larger and larger species at an increasingly better level of theory. The first studies focused on the identification of small species by comparison between their theoretical and experimental vibrational fingerprints. Starting from aromatic natural amino acids which possess a convenient UV chromophore (see Sect. 2), many seminal studies have been devoted to these natural model peptides. Their biological relevance can be questioned, in particular because the N and C natural terminals adopt a zwitterionic structure under physiological conditions which is usually not the structure observed in the gas phase. These natural peptides nevertheless constitute handy model systems on which experimental techniques and theoretical methodologies have been refined. This approach became increasingly difficult as the targeted systems were larger and larger. Among these, capped peptides, whose N and C terminals were made of complete peptide bonds, enabled progress to be made towards greater biological relevance. Successful investigation of systems as large as tripeptides stemmed from a gradual knowledge of species of increasing size, following the so-called bottom-up approach. A set of essential secondary structures has been identified, demonstrating the biological relevance of such gas-phase studies. By demonstrating that relevant models mimicking the backbone features of proteins could be studied in the gas phase at an unprecedented level of accuracy, these pioneering works opened new horizons and stimulated a large community. Taking advantage of decisive quantum chemistry progress, a more thorough approach appeared where a global theoretical study of the PES was conducted. Close comparison between the experimentally observed landscape and its theoretical counterpart provides much richer outcomes, allowing a fruitful interplay between experiment and theory. A comprehensive and precise PES enables a reliable assignment of the species observed, including all minor conformers. In return, failures of quantum chemistry methods to reproduce experimental observations could be spotted, guiding further theoretical developments. The aim of this review is first to describe the parallel advancements in both experimental and theoretical methods and procedures, together with the crossfertilisation that resulted. Examples of striking secondary structures of proteins isolated in the gas phase are given. Highlights on specific shaping interactions, including intermolecular interactions, e.g. with the solvent, provide an in-depth understanding of protein structure. In a last part, it is shown how conformationselective spectroscopy is able to document the diversity of the PES landscape of
Isolated Neutral Peptides
229
these species, thus revealing their flexibility. It is also shown how coupling IR and UV information enables spectroscopists to target conformer-selective photophysics in peptides. Finally, future prospects of gas phase conformation-selective spectroscopy are presented.
2 Two Synergetic Approaches 2.1
Experiment
Vibrational spectroscopy, based on IR or Raman absorption, is an efficient technique to identify the conformational structure of small molecules, including model peptide systems [22–33]. It is, however, confronted by spectral congestion when dealing with molecules of increasing flexibility with a pool of conformations which interconvert at room temperature. Advanced procedures are required to gain valuable conformation-specific spectroscopic information. An efficient cooling usually provided by a supersonic expansion is needed to remove the room temperature internal energy, typically 3.103 cm1 in a capped amino-acid. In addition, techniques such as temperature- or concentration-dependence studies are needed to identify the contributions of conformers and clusters [23, 27, 28, 30]. Several conformer-selective experiments were proposed, such as those based on a strong field deflection [42]. In this context, IR/UV double resonance experiments [43, 44] became quite popular. When conformer-selective excitation of a UV chromophore is possible, these experiments provide spectroscopists with an elegant way to record conformer-selective IR spectra [45]. Combined with classical thermal vaporisation techniques [46, 47] or with more sophisticated laser-desorption-based techniques [48–50] coupled to a supersonic expansion, IR/UV experiments have dramatically boosted the research field pioneered by Levy’s group [48]. They made possible the observation of vibrationally resolved IR spectra (Fig. 1), first from the NH stretch spectroscopy permitted by the development of table-top OPOs in this range [45]. From the basic principles of IR spectroscopy, H-bonding in jet-cooled peptides can be reliably identified, providing a first basis to conformational assignment, further refined by comparison with quantum chemistry calculations. Rapidly, several conformers of the three natural amino-acids Phe [16], Trp [20] and Tyr [56], and sometimes their hydrates [57, 58], were identified through their vibrational fingerprint, refining former pioneering studies based on UV spectroscopy only [48, 59–62]. Numerous studies on peptides containing one of the three natural aromatic residues followed [46, 47, 51–55, 63–118], the presence of a near-UV chromophore being the tribute to pay to gain the conformer-selectivity brought by the IR/UV double resonance technique. Non-natural residues derived from the natural UV chromophores have also been widely employed [52, 111, 119– 129]. Beyond this constraining aspect, the UV frequency and FC pattern being
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sensitive to the interactions involving the aromatic ring, they can provide very useful structural clues (Sect. 4.2). Of course, the principle of the experiment relies on the conformational selectivity of the UV chromophore. This is made possible by the intrinsic sensitivity of the UV transition to the chromophore environment, the cooling achieved in the expansion enabling the detection of frequency differences as small as a few cm1, in practice limited by the rotational contour of the UV bands. This selectivity can in general be achieved for small species, apart from spectral coincidence cases, which require a more sophisticated treatment [130]. In larger species, the spectral broadening caused by the difficulty in evacuating the internal energy results in a lack of UV resolution which affects the conformational selectivity [54, 89, 107, 108]. In these cases, one has to rely on the interpretation of the IR spectrum to figure out whether conformational selectivity is achieved or whether several species are simultaneously present. It should be noted that sequences deprived of UV chromophore can also been investigated by introducing so-called UV-tags which can be covalently [105, 107, 127, 131–145] or non-covalently bound, as in peptide-toluene complexes (Gloaguen E, Mons M, unpublished results). The assumption that the desired UV chromophore (often a phenyl ring) is weakly interacting with the rest of the molecule turned out to be a relatively seldom situation, owing to the huge flexibility of the peptides. Biases are introduced by what can be seen as parasitic interactions, e.g. NH-π [131], which prevent to extract any intrinsic data about the untagged system, especially in terms of conformational energetics. Gas phase peptides of various sizes have been investigated in several spectral ranges (Fig. 1a–e), the first of them, in both importance (number of studies), reliability and interpretative potentialities is probably the so-called amide A NH-stretch region (Fig. 1c, d). The NH stretch oscillator exhibits an exquisite sensitivity to its environment, and is virtually uncoupled with the potentially numerous other NH groups of the molecules. Then, each individual IR band bears qualitative information about the strength of the H-bond in which the corresponding NH group is engaged [34], apart from very specific cases where strong hyperconjugation effects involve the σ* orbital of the vibrational probe [146]. Moreover, other vibrational probes lie in the amide A NH-stretch region, and contribute to make this spectral range very popular: amine NH and carboxylic acid OH for natural peptides, indole NH fot Trp, imidazole NH for His, phenol OH for Tyr, alcohol OH for Ser and Thr as well as water OH in microhydrated peptides. For the simplest systems, the H-bonding content can be deduced just by “reading” the conformer-selective amide A spectrum. Spectroscopic tricks, such as spectral correlations between physically coupled NH oscillators in NH2-capped C-terminal groups [34, 70], together with the analysis of UV spectroscopic features (see Sect. 4.2), provide additional structural clues which have enabled the tackling of more complex species. Phe-containing dipeptides have been confidently assigned without relying on quantum calculations, and a ranking of the H-bonds according to their strength has been proposed (C5
