compatibility with native protein structures and effects on protein–protein interactions

Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids—aliphatic, aromatic, charged and polar as well as proline—is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references)

Biophysikalische Charakterisierung der Wechselwirkungen einzelner fluorhaltiger Aminosäuren innerhalb einer nativen Polypeptidumgebung

Despite its low abundance in naturally occurring biomolecules, fluorine’s often favorable impact on their pharmacokinetic and biological properties makes it an outstanding element. In fact, fluorination has become a standard tool in pharmaceutical lead optimi-zation. Peptides and proteins carrying fluorine as an amino acid side chain substituent have also been the topic of numerous studies in recent years. These investigations have revealed interesting results concerning the membrane permeability, structural stability, and stability towards proteolysis of some biologically relevant peptides. However, our current understanding of how these building blocks affect structure formation and thus the biological activity of peptides and proteins is limited. Therefore, it remains difficult to predict the effects of side chain fluorination within natural protein environments. Thus, a complete characterization of the molecular interactions of fluorinated amino acids with their native counterparts in the context of protein-protein interactions is necessary. In line with the present thesis the hydrophobicity of different analogues of (S)-2-aminobutyric acid with increasing fluorine content and side chain volume was investigated by applying an HPLC assay. Furthermore, a newly designed peptide model based on the alha-helical coiled coil was used to characterize the heterodimerization of different monomers that present these amino acids within the hydrophobic core with an exclusively native complement. The amino acids were incorporated at two specified positions (either a16 or d19) within the hydrophobic core. With support from theory, the effects on coiled-coil structure and stability were studied by applying CD spectroscopy. It was shown that the impact of size and polarity of the fluorinated building blocks highly depends on the immediate environment of the substitution within the hydrophobic core. At position a16, stability is mainly determined by side chain volume. At position d19, however, the destabilizing impact of fluorine- induced polarity, due to the distinctly different orientation of the side chains at this position, prevails. The impact of fluorination on coiled-coil folding kinetics was studied by applying a surface plasmon resonance-based biosensor. These investigations revealed that variations in fluorine content mainly affect the association of the monomers, since it highly depends on their hydrophobicity. As has been recognized before, the kinetic data indicate that high fluorine content, despite increasing hydrophobicity, apparently retards association. Whether this effect is based on the formation of ‘fluorous clusters’ that stabilize the unfolded state of the monomers cannot be concluded at present.Trotz seiner geringen Häufigkeit als Substituent in natürlichen vorkommenden Biomo-lekülen ist Fluor auf Grund seines positiven Einflusses auf die pharmakokinetischen und biologischen Eigenschaften von Wirkstoffen ein außerordentlich interessantes Element. Diese Eigenschaften machen die Fluorierung heute zu einem Standard in der Optimierung pharmakologischer Leitstrukturen. Auch fluorierte Aminosäuren wurden in den vergangenen Jahren in zahlreichen Studien untersucht. Diese Studien zeigen, dass der Einbau von Fluor durchaus positive Effekte beispielsweise auf die Membranpermeabilität, die Strukturstabilität sowie die Stabilität gegenüber Proteasen einiger biologisch relevanter Peptide hat. Jedoch ist das aktuelle Verständnis der Eigenschaften fluorierter Aminosäuren noch begrenzt; das heißt, dass es bis heute nicht ohne weiteres möglich ist, den Einfluss der Fluorierung in der Umgebung nativer Proteine vorauszusagen. Eines der attraktivsten Ziele der biologischen Fluorchemie ist daher eine komplette Charakterisierung der molekularen Wechselwirkungen fluorierter Aminosäuren mit nativen Seitenketten im Kontext von Protein-Protein-Wechselwirkungen. Im Rahmen dieser Arbeit wurden fluorierte Analoga der Aminosäure (S)-2-aminobuttersäure hinsichtlich ihrer Hydrophobie mittels HPLC untersucht. Unter Verwendung eines neu entworfenen Proteinmodells auf Basis des alpha-helikalen Coiled Coil Faltungsmotivs wurde weiterhin die Heterodimerisierung verschiedener Peptide, die diese Aminosäuren im hydrophoben Kern enthalten, mit einem ausschließlich nativen komplementären Partnerpeptid charakterisiert. Die Aminosäuren wurden an zwei ausgewiesenen Stellen (a16 oder d19) eingebaut, um ihre Effekte auf die Struktur und Stabilität der Coiled Coils mittels CD Spektroskopie zu untersuchen. Mit Unterstützung durch theoretische Berechnungen wurde gezeigt, dass sich Unterschiede in Volumen und Polarität der fluorierten Seitenketten hinsichtlich ihrer Effekte auf die Stabilität in Abhängigkeit von ihrer unmittelbaren Umgebung im hydrophoben Kern unterscheiden. Während in Position a16 das Volumen der Seitenketten die Stabilität des Coiled Coils bestimmt, überwiegt in Position d19 auf Grund der unterschiedlichen Orientierung der Seitenketten der destabilisierende Einfluss fluorinduizerter Polarität. Der Einfluss der Fluorierung auf die Kinetik der Faltung des Coiled Coils wurde mittels Surface Plasmon Resonance untersucht. Hier zeigte sich, dass Änderungen im Fluorierungsgrad hauptsächlich die Assoziation der Monomere beeinflusst, da diese erheblich von der Hydrophobie abhängt. Wie bereits in früheren Studien gezeigt, scheint ein hoher Fluorierungsgrad trotz erhöhter Hydrophobie die Assoziation zu verlangsamen. Ob dieser Effekt auf der Ausbildung von „Fluorclustern“ im ungefalteten Zustand der Monomere beruht, kann zu diesem Zeitpunkt jedoch nicht eindeutig nachgewiesen werden

Position-Dependent Effects of Fluorinated Amino Acids on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil

Systematic model investigations of the molecular interactions of fluorinated amino acids within native protein environments substantially improve our understanding of the unique properties of these building blocks. A rationally designed heterodimeric coiled coil peptide (VPE/VPK) and nine variants containing amino acids with variable fluorine content in either position a16 or d19 within the hydrophobic core were synthesized and used to evaluate the impact of fluorinated amino acid substitutions within different hydrophobic protein microenvironments. The structural and thermodynamic stability of the dimers were examined by applying both experimental (CD spectroscopy, FRET, and analytical ultracentrifugation) and theoretical (MD simulations and MM-PBSA free energy calculations) methods. The coiled coil environment imposes position-dependent conformations onto the fluorinated side chains and thus affects their packing and relative orientation towards their native interaction partners. We find evidence that such packing effects exert a significant influence on the contribution of fluorine-induced polarity to coiled coil folding.publishe

Hyperosmotic Infusion and Oxidized Surfaces Are Essential for Biofilm Formation of Staphylococcus capitis From the Neonatal Intensive Care Unit

Staphylococcus capitis is an opportunistic pathogen often implicated in bloodstream infections in the neonatal intensive care unit (NICU). This is assisted by its ability to form biofilms on indwelling central venous catheters (CVC), which are highly resistant to antibiotics and the immune system. We sought to understand the fundamentals of biofilm formation by S. capitis in the NICU, using seventeen clinical isolates including the endemic NRCS-A clone and assessing nine commercial and two modified polystyrene surfaces. S. capitis clinical isolates from the NICU initiated biofilm formation only in response to hyperosmotic conditions, followed by a developmental progression driven by icaADBC expression to establish mature biofilms, with polysaccharide being their major extracellular polymer substance (EPS) matrix component. Physicochemical features of the biomaterial surface, and in particular the level of the element oxygen present on the surface, significantly influenced biofilm development of S. capitis. A lack of highly oxidized carbon species on the surface prevented the immobilization of S. capitis EPS and the formation of mature biofilms. This information provides guidance in regard to the preparation of hyperosmolar total parenteral nutrition and the engineering of CVC surfaces that can minimize the risk of catheter-related bloodstream infections caused by S. capitis in the NICU